传统光学相干断层成像:软骨双折射特性的研究

传统光学相干断层成像可实现无损伤在体检测,具有较高的分辨率和灵敏度。获取生物组织的双折射信息可望是其应用之一。本文用传统光学相干断层成像系统以动物模型对关节软骨进行了研究,对软骨组织的传统光学相干断层图像的层结构随脱水和机械作用的变化进行了分析。结果表明,传统光学相干断层成像系统在一定程度上可用于监测由其胶原质纤维排列决定的软骨组织的双折射和密度。     

激光共聚焦显微技术在瓮安生物群中的应用

激光共聚焦显微技术是一种以激光作为激发光源,通过特殊装置"针孔"来过滤离焦光线以提高光学分辨率和对比度的光学成像技术。由于大部分化石不能自发荧光,该技术在古生物学领域尚未实现大范围的应用。但若围岩能自发荧光而与化石之间具有一定衬度,或化石因含特殊成分能在特定波段激光照射下自发荧光而产生结构衬度,则可以运用激光共聚焦显微技术获得在普通光学显微镜及荧光显微镜下难以清晰观察到的信息。为推动激光共聚焦技术在古生物学领域中的应用,文中系统介绍了该技术的原理与使用方法,并以埃迪卡拉纪磷酸盐化特异埋藏的瓮安生物群微体化石为例,展示了该技术在化石成像中的若干优势。实验结果表明,瓮安生物群微体化石因富含磷灰石可自发荧光实现成像,使用激光共聚焦显微成像技术观察瓮安生物群化石薄片不仅可以获得较好衬度,而且还能提高成像的分辨率和清晰度。此外,在化石薄片的厚度范围内还可以实现化石结构三维重建。     

二次谐波显微成像技术

二次谐波非线性显微成像技术是近年发展起来的一种新型光学成像方法,已广泛应用于生物医学的各个领域。介绍了光学二次谐波产生的原理、成像装置及其技术发展,描述了二次谐波的成像特点和它与双光子荧光成像的异同,并对其在生物医学上的应用及发展前景做出展望。     

光学相干断层成像在临床医学中的应用

光学相干断层成像(OCT)是一种基于弱相干光学断层成像技术,可以对生物组织活体断层成像,是继计算机X射线摄影(CR)和数字X射线摄影(DR)、超声、电子计算机断层扫描(CT)、磁共振成像(MRI)之后又一新的生物组织成像方法。OCT在眼科、皮肤科、心血管科、肿瘤科、骨科、口腔科、妇科等对组织病变的早期光学诊断和实时动态监测方面具有广泛的应用及重要的临床价值。本文就OCT的基本原理、研究现状、主要的临床应用和应用过程中存在的问题进行综述并展望未来相关的发展趋势。     

GFP标记的肿瘤生长和转移的整体荧光成像

Fugene 6脂质体介导pEGFP-C1转染人源肺癌细胞(SPC-A1),经G418抗性筛选和96孔板有限稀释获得稳定高表达GFP的单克隆细胞株SPC-A1-EGFP。裸鼠腹腔注射SPC-A1-EGFP细胞建立自发转移模型;裸鼠尾静脉注射SPC-A1-EGFP细胞建立实验转移模型。利用整体光学成像系统(wllole-body optical imaging system)对荷瘤鼠整体荧光成像。结果表明,整体光学成像系统可实时非侵入监测腹腔肿瘤生长和扩散过程,通过胸腔皮瓣窗chest—wall skin-flap window)可低侵入检测肺转移。该研究为在体监测原位移植瘤的自发转移和发现抗肿瘤新药物提供了良好实验平台。     

光学神经成像研究进展

大脑功能的成像检测在认知神经科学领域具有极其重要的意义。现代光子学技术的发展为认知脑成像提供了新的研究手段,在神经系统信息处理机制研究中发挥重要作用。文章介绍了在神经元、神经元网络、特定脑皮层功能构筑以及系统与行为等不同层次开展神经系统信息处理机制研究的各种光学成像技术,包括多光子激发荧光显微成像、内源信号光学成像、激光散斑成像和近红外光学成像等,并评述了这些有特色的光学成像技术在多层次获取和分析神经信息中的研究进展。     

三维X射线显微技术与小型-微体化石高分辨率无损成像*

在古生物学研究中,以X射线断层成像(Computed Tomography)为代表的三维无损成像技术可以在不破坏化石标本的前提下,同时获得标本外观形态和内部结构的信息,相比传统的可见光成像手段有着明显优势。为推动化石三维无损成像技术在国内古生物学领域的发展,本文系统介绍一种新型显微CT技术——三维X射线显微术(Three-Dimensional X-ray Microscopy)。与基于几何放大和吸收衬度成像的传统显微CT技术相比,该技术有若干优势:(1)将同步辐射X射线显微断层成像的光学成像系统引入基于实验室X射线源的显微CT系统中,在几何放大的基础上增加了光学放大,优化了传统显微CT的系统架构,弥补了传统显微CT单纯依靠几何放大的不足,提高了空间分辨率;(2)采用可移动的X射线源和优化的光学成像系统,实现了低能X射线相位衬度成像,可以三维重构传统显微CT技术无法有效探测的、低吸收衬度的化石标本;(3)基于新的成像架构和成像算法,实现了厘米-分米级较大标本内部"感兴趣区域"(Region of Interest)精确导航和局部高分辨率(微米-亚微米空间分辨)成像;(4)可以实现小型扁平标本(宽厚比4,宽10cm)高效率、高分辨率成像和长条形微体标本长轴方向自动分段无缝拼接的微米至亚微米级高分辨率重建,弥补了传统工业显微CT针对小型扁平标本和长条形微体标本高分辨成像效果不佳的缺陷。这些优势使得基于实验室X射线源的显微CT成像技术可以获得接近同步辐射X射线源的成像质量,从而有效推动化石生物学研究。     

适用于细胞荧光测量的多道显微荧光计

建立了由倒置荧光显微镜和光学多道分析仪(OMA)连接而组成的适用于细胞荧光测量的多道显微荧光计,编制了数据处理程序。利用这一装置测量了单个细胞,多细胞的荧光光谱和拓扑(topography)。和传统的显微荧光计相比,该装置具有测量灵敏度和精度高、速度快等特点,可用来进行活细胞动态过程的研究。     

微波热声实时成像系统及应用

本文建立了一套微波热声实时成像系统,该系统由脉冲微波发生器,多元环形探测器,多通道数据采集装置和数据重建装置共同组成。在实验中,利用填充盐水的两个塑料管验证其实时成像的性能,结果表明,该系统能够实现每秒16.7帧的成像速度。随后,对活体小鼠的正常区域和肿瘤区域分别进行热声成像,得到肿瘤和正常区域的对比度为1.7∶1,证明了该系统在肿瘤检测中有较高的对比度。最后,利用该系统监控细管趋近离体肿瘤的过程。因此该系统有望应用于实时监测。综上所述,该热声成像系统具有无损,成像速度快和大视场的良好性能,有望在生物医学中得到广泛的应用,尤其在肿瘤筛查和实时监控方面发挥作用。     

β淀粉样蛋白斑块的高分辨全脑三维定量研究

中枢神经系统中β淀粉样蛋白斑块是阿尔兹海默症的主要病理特征之一,其负荷和数目的变化是病程发展的重要标志.已有研究主要是对局部脑组织进行二维切片成像,尚缺少在全脑三维空间对斑块进行高分辨率定量分析的研究方法.本文建立了适用于哺乳动物三维完整脑内β淀粉样蛋白斑块定量分析策略,包括全脑斑块快速荧光染色方法、基于荧光显微光学切片断层成像技术的高分辨全脑数据获取,以及斑块自动定位、统计数目等.与免疫组化染色比较,证明本方法对直径大于10μm的斑块检出率为97.71%±0.18%.并以0.32μm×0.32μm×2μm的成像分辨率,获取了5XFAD转基因小鼠全脑Aβ斑块分布数据集,首次以脑区/核团的三维轮廓划分出立体区域,定量统计了90个亚区内Aβ斑块的数量及分布密度.本文建立的快速、精准、价廉的方法将有助于全面高效地研究阿尔兹海默症致病机理和药效评估.     

Real-time tracking of root hair nucleus morphodynamics using a microfluidic approach

Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RHs show a fast apical growth, constituting a unique single cell model system for analysing cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, although high-resolution imaging is still lacking to enable an investigation of the accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) that enables us to capture high resolution confocal imaging of Arabidopsis RH development with real-time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, to illustrate the possibilities offered by the CMD, we have compared the real-time variations in the circularity, area and aspect ratio of nuclei moving in growing and mature RHs. Interestingly, we observed higher aspect ratios in the nuclei of mature RHs, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development.     

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo^1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest^3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells⁵, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium⁶, cAMP^7-8, inositol phosphates⁹ and cGMP^10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.     

Design of an MRI-compatible robotic stereotactic device for minimally invasive interventions in the breast

The objective of this work was to develop a robotic device to perform biopsy and therapeutic interventions in the breast with real-time magnetic resonance imaging (MRI) guidance. The device was designed to allow for (i) stabilization of the breast by compression, (ii) definition of the interventional probe trajectory by setting the height and pitch of a probe insertion apparatus, and (iii) positioning of an interventional probe by setting the depth of insertion. The apparatus is fitted with five computer-controlled degrees of freedom for delivering an interventional procedure. The entire device is constructed of MR compatible materials, i.e. nonmagnetic and non-conductive, to eliminate artifacts and distortion of the MR images. The apparatus is remotely controlled by means of ultrasonic motors and a graphical user interface, providing real-time MR-guided planning and monitoring of the operation. Joint motion measurements found probe placement in less than 50 s and sub-millimeter repeatability of the probe tip for same-direction point-to-point movements. However, backlash in the rotation joint may incur probe tip positional errors of up to 5 mm at a distance of 40 mm from the rotation axis, which may occur for women with large breasts. The imprecision caused by this backlash becomes negligible as the probe tip nears the rotation axis. Real-time MR-guidance will allow the physician to correct this error Compatibility of the device within the MR environment was successfully tested on a 4 Tesla MR human scanner     

Real-time monitoring of the strand displacement amplification (SDA) of human cytomegalovirus by a new SDA-piezoelectric DNA sensor system

Nucleic acid amplification has long been used in biosensor technologies, such as DNA sensors, DNA chips and microarrays, due to its advantage of high sensitivity in detecting target DNA. However, dynamic monitoring of nucleic acid amplifications with traditional DNA sensors in real-time is difficult since a constant temperature must be maintained during detection. Thus, the piezoelectric sensor, one type of traditional DNA sensor, is not applicable in real-time monitoring PCR due to the dramatic change in temperature that occurs during reaction. In this study, we introduced strand displacement amplification (SDA), an well-developed nucleic acid amplification technique that can work under conditions of constant temperature, into the development of a novel piezoelectric sensor. Using the new SDA-piezoelectric DNA sensor, we designed a stable system for liquid-phase detection, in which the crystal oscillator plate was fixed by an easily adjustable screw-threaded clamping mechanism and successfully applied the new sensor system to real-time SDA monitoring of human cytomegalovirus (HCMV). This new technique overcomes the shortcomings of traditional DNA sensors in real-time monitoring of nucleic acid amplification. The technique has proved to be a markedly simplified procedure with a number of advantages, such as higher sensitivity, better time efficiency, and the ability of dynamic real-time detection.     

Quantitative bioluminescence imaging of tumor-targeting bacteria in living animals

We describe a protocol for imaging bacterial luciferase (Lux)-expressing bacteria in small living animals. In this protocol, light emitted by Lux-expressing bacteria is detected and monitored by a cooled charge-coupled device detector. When these bacteria are administered to animals, it provides a potentially valuable approach to generate sensitive whole-body images with extremely low background. This imaging technology should enable the real-time monitoring of bacterial migration into both primary and metastatic tumors in several different mouse tumor models at a strong quantification power.     

Transducer for monitoring respiration during imaging procedures

A transducer system for monitoring respiration is described; it uses a ‘liquid column’ sensor with a remote integrated circuit pressure module. It was designed primarily for non-invasive monitoring and control of respiration during diagnostic imaging procedures, but has also found applications in other areas, e.g. physiotherapy and pulse monitoring. The device is a new version of a system developed several years ago and takes advantage of relatively low cost commercial ‘building blocks’. The output is an analogue voltage (from a low impedance source) capable of driving a wide range of recorders, amplifiers and computer interfaces. Reference is also made in the text to a bio-feedback signal processing and display unit (described elsewhere) which, when used with this transducer, provides a versatile respiratory control system.     

基于傅里叶变换近红外光谱实时分析1,3-丙二醇发酵过程生物量的在线监测方法

生物量是反映生物发酵过程进展的重要参数,对生物量进行实时监测可用于对发酵过程的调控优化。为克服目前主要采用的离线方法检测生物量时间滞后和人工测量误差较大等缺点,本研究针对1,3-丙二醇发酵过程设计了一个基于傅里叶变换近红外光谱实时分析技术的生物量在线监测实验平台,通过对实时采集光谱预处理以及敏感光谱段分析,应用偏最小二乘算法,建立了1,3-丙二醇发酵过程生物量变化的动态预测模型。以底物甘油浓度为60 g/L和40 g/L的发酵过程作为外部验证实验,分析得到模型的预测均方根误差分别为0.341 6和0.274 3,结果表明所建立的模型具有较好的实时预测能力,能够实现对1,3-丙二醇发酵过程中生物量的有效在线监测。     

Monitoring Radiofrequency Ablation Using Real-Time Ultrasound Nakagami Imaging Combined with Frequency and Temporal Compounding Techniques

Gas bubbles induced during the radiofrequency ablation (RFA) of tissues can affect the detection of ablation zones (necrosis zone or thermal lesion) during ultrasound elastography. To resolve this problem, our previous study proposed ultrasound Nakagami imaging for detecting thermal-induced bubble formation to evaluate ablation zones. To prepare for future applications, this study (i) created a novel algorithmic scheme based on the frequency and temporal compounding of Nakagami imaging for enhanced ablation zone visualization, (ii) integrated the proposed algorithm into a clinical scanner to develop a real-time Nakagami imaging system for monitoring RFA, and (iii) investigated the applicability of Nakagami imaging to various types of tissues. The performance of the real-time Nakagami imaging system in visualizing RFA-induced ablation zones was validated by measuring porcine liver (n = 18) and muscle tissues (n = 6). The experimental results showed that the proposed algorithm can operate on a standard clinical ultrasound scanner to monitor RFA in real time. The Nakagami imaging system effectively monitors RFA-induced ablation zones in liver tissues. However, because tissue properties differ, the system cannot visualize ablation zones in muscle fibers. In the future, real-time Nakagami imaging should be focused on the RFA of the liver and is suggested as an alternative monitoring tool when advanced elastography is unavailable or substantial bubbles exist in the ablation zone.     

Short-term BOD (BODst) as a parameter for on-line monitoring of biological treatment process; Part II: instrumentation of integrated flow injection analysis (FIA) system for BODst estimation

An instrument with integrated flow injection analysis (FIA) system has been developed for on-line monitoring a process for conversion of biomass under field condition. The instrument consists of a newly designed biosensor for easy renewal of the bio-receptor without disassembling the sensor, a FIA controller for controlling the analysis operations, and a computer-based data acquisition system for data recording and processing. The instrument performed a sequence operations automatically including preparation of sample in the desired concentration, sample loading, sample injection, signal recording, data processing, and self-cleaning of the system. This makes the instrument being an interesting and promising device for on-line process monitoring.