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Widefield heterodyne interferometry with a novel custom 2D CMOS camera for biological imaging 宽视场外差干涉测量与一种新的定制的二维CMOS相机的生物成像
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154836
Jing Zhang, R. Light, Nicholas S. Johnston, M. Somekh
Widefield interferometry is a useful technique for the fast visualisation of low contrast objects. In addition, the spatial resolution of images is improved and quantitative measurement becomes possible with this technique.
宽视场干涉测量是一种有效的低对比度物体快速可视化技术。此外,该技术还提高了图像的空间分辨率,使定量测量成为可能。
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引用次数: 0
Optimisation of plasmonic-based biosensors for label-free imaging of molecular arrays; towards diagnosis of neurodegenerative diseases 分子阵列无标记成像等离子体生物传感器的优化研究神经退行性疾病的诊断
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154819
J. Richens, K. Vere, P. O'shea
Plasmonic-based biosensors offer huge potential as diagnostic instruments. The greatest benefit of such detection modalities is that they are label-free and thus only a single selective ligand is required for target detection, decreasing both protocol time and the required user proficiency. However, during technology development, the problems associated with using a detection system based upon an addition of mass need to be considered to ensure accurate target detection and quantification. This is particularly important in diagnostic applications as biomarkers are likely to be identified in complex bodily fluids including blood plasma, cerebrospinal fluid or saliva. We have extended these analytical approaches by implementing molecular arrays that recognise panels of marker biomolecules to discriminate the presence and severity of Alzheimer's Disease.
等离子体生物传感器作为诊断仪器具有巨大的潜力。这种检测方式的最大好处是它们是无标签的,因此只需要一个单一的选择性配体来检测目标,减少了协议时间和所需的用户熟练程度。然而,在技术开发过程中,需要考虑使用基于质量加法的检测系统的相关问题,以确保准确的目标检测和量化。这在诊断应用中尤为重要,因为生物标志物很可能在复杂的体液中被识别,包括血浆、脑脊液或唾液。我们已经扩展了这些分析方法,通过实施分子阵列来识别标记生物分子面板,以区分阿尔茨海默病的存在和严重程度。
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引用次数: 1
The effect of encapsulation on molecular computing efficiency 包封对分子计算效率的影响
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154848
J. Chaplin, N. Krasnogor, Noah A. Russell
Research into molecular computation offers exciting possibilities for interfacing computation with biological systems. This could be achieved using light to switch photochromic molecules between states. For example, 6-Nitro-BIPS2 can be switched from a Spiropyran (SP) state to a Trans-Merocyanine (MC) state using UV photons while visible light switches from MC to SP. The MC state is also fluorescent. Modified spiropyrans targeted to proteins can improve imaging contrast3, alter enzyme activity4, alter protein interactions5 and switch vesicle permeability6.
分子计算的研究为将计算与生物系统相结合提供了令人兴奋的可能性。这可以通过光在不同状态之间切换光致变色分子来实现。例如,6-硝基bips2可以利用紫外光子从螺吡喃(SP)状态切换到反式merocyanine (MC)状态,而可见光则从MC状态切换到SP状态。MC状态也是荧光的。靶向蛋白质的修饰螺吡喃类蛋白可以改善成像对比度、改变酶活性、改变蛋白质相互作用和改变囊泡通透性。
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引用次数: 0
Multimodal imaging of fluid transport in living epithelial sheets 活体上皮细胞内液体运输的多模态成像
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154825
K. Webb, Jing Zhang, M. Somekh
Epithelial tissues form the barrier between different fluid compartments throughout the body, lining and delineating the borders between tissues to control homeostasis and provide for the functions of secretion, absorption, and volume regulation. Key to these physiological roles is the transepithelial transport of fluid and solutes. Vectorial transport is possible due to the highly polarised cytoarchitecture, with different ion transporters and other proteins present in the apical vs basolateral membrane domains, which are separated by “tight junctions” which ring the lateral membranes of each cell. These separate the apical and basolateral compartments, providing intercellular adhesion and controlling permeation via the intercellular pathway. By varying the composition and function of the pools of transport proteins between these segregated membrane domains, directed transport is achieved thus promoting homeostasis. The retinal pigment epithelium (RPE) lies behind the neural retina, forming the blood-retinal barrier and providing for the homeostasitic and biochemical support of the photoreceptors and other neuronal layers. Much is known of RPE physiology at the macroscopic level since dysregulation or pathology have profound consequences for the visual system. Lacking is the detailed knowledge of biophysical mechanisms and local intercompartmental dynamics by which fluid and solute transport is achieved and regulated at the cellular and subcellular level.
上皮组织在全身不同的液体室之间形成屏障,在组织之间形成边界,控制体内平衡,并提供分泌、吸收和体积调节功能。这些生理作用的关键是液体和溶质的经上皮运输。载体运输是可能的,因为高度极化的细胞结构,不同的离子转运体和其他蛋白质存在于顶端和基底外侧膜域,它们被“紧密连接”分开,这些“紧密连接”环绕着每个细胞的外侧膜。它们将细胞顶室和基底外侧室分开,提供细胞间粘附并通过细胞间通路控制渗透。通过改变这些分离膜结构域之间转运蛋白池的组成和功能,实现了定向转运,从而促进了体内平衡。视网膜色素上皮(RPE)位于神经视网膜后面,形成血视网膜屏障,为光感受器和其他神经元层提供稳态和生化支持。由于失调或病理对视觉系统有深远的影响,因此在宏观水平上对RPE生理学有很多了解。缺乏生物物理机制和局部室间动力学的详细知识,通过这些机制,流体和溶质运输在细胞和亚细胞水平上实现和调节。
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引用次数: 0
Custom CMOS cameras for configurable and adaptive imaging 定制CMOS相机可配置和自适应成像
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154831
R. Light
Many scientific optical measurement problems require specialised processing, examples are phase stepping, pump probe experiments or speckle averaging. Many of these are carried out with a single photodetector or with conventional cameras. The ability to integrate custom electronics alongside photodetectors allows custom cameras to be developed that are well suited to these specialised techniques. These can provide on chip processing and adaptive responses to the incoming signals.
许多科学光学测量问题需要专门的处理,例如相位步进,泵浦探针实验或散斑平均。其中许多都是用单个光电探测器或传统相机完成的。将定制电子设备与光电探测器集成在一起的能力使定制相机能够开发出非常适合这些专业技术的定制相机。这些可以提供片上处理和自适应响应输入信号。
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引用次数: 0
Optical sectioning and fast optical focussing in microscopy 显微镜中的光学切片和快速光学聚焦
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154833
T. Wilson
The fundamental property, which any optical microscope that is to be used to finally produce three-dimensional images of a volume specimen must possess, is the ability to image efficiently (and infocus) only those regions the specimen that lie within a thin section in the focal region of the microscope. In order to image a three-dimensional volume of a thick specimen it is necessary to take a whole series of such thin optical sections as the specimen is moved axially through the focal region. There are many methods to produce optical sectioning of which the confocal optical system is just one. We shall review these methods and describe a particularly convenient method of implementation that uses white light illumination and real-time image formation and can lead, amongst other things, to enhanced optical sectioning.
任何用于最终产生体积标本三维图像的光学显微镜都必须具备的基本特性是能够有效地成像(和聚焦)位于显微镜聚焦区域的薄片内的标本的那些区域。为了成像一个厚标本的三维体积,有必要采取整个系列的这样的薄光学切片的标本轴向移动通过焦点区域。产生光学切片的方法有很多,共聚焦光学系统只是其中一种。我们将回顾这些方法,并描述一种特别方便的实现方法,该方法使用白光照明和实时图像形成,并且可以在其他方面导致增强光学切片。
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引用次数: 0
A custom CMOS camera for sensitive SPR measurement 一个定制的CMOS相机敏感的SPR测量
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154832
R. Light, Richard J. Smith, Nicholas S. Johnston, M. Somekh
In surface plasmon imaging, a sample is placed on a glass prism that has been coated with a metal film, usually gold. When illuminated at the appropriate angle, plasmons are generated in the gold surface and there is both a sharp dip in the reflectivity of the gold and a strong phase shift in the reflected light. The plasmon angle is very sensitive to the conditions on the gold surface, which makes it potentially very useful in label-free biological measurements, where the amount of a particular protein in a sample can be determined by how much binds to antibodies printed on the gold surface and changes the local surface properties.
在表面等离子体成像中,样品被放置在涂有金属膜(通常是金)的玻璃棱镜上。当以适当的角度照射时,在金表面产生等离子体激元,并且金的反射率急剧下降,反射光中有强烈的相移。等离子体角度对金表面的条件非常敏感,这使得它在无标签生物测量中非常有用,其中样品中特定蛋白质的数量可以通过与印在金表面的抗体结合的多少来确定,并改变局部表面特性。
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引用次数: 0
Advances in time-resolved fluorescence microscopy: Simultaneous FRAP, FLIM and tr-FAIM to image rotational and translation diffusion in living cells 时间分辨荧光显微镜的进展:FRAP, FLIM和tr- fam同时成像活细胞的旋转和平移扩散
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154838
K. Suhling, P. Chung, J. Levitt
Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are minimally invasive and can be applied to live cells and tissues. It is advantageous to exploit the many properties of fluorescence in imaging experiments.[1–3] We demonstrate a novel experimental arrangement for measurements of intracellular dynamics by simultaneous acquisition of fluorescence recovery curves (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). We have used this set-up to obtain the translational and rotational diffusion properties of green fluorescent protein (GFP)-labelled proteins in living cells. This method allows extraction of fluorescence lifetimes, rotational correlation times and diffusion characteristics simultaneously and thus avoids excessive photobleaching or artefacts due to cell movement. It can also measure phenomena that each method on its own cannot measure, e.g. diffusing homo-dimers.
荧光成像技术是生物和生物医学科学的有力工具,因为它们是微创的,可以应用于活细胞和组织。这有利于在成像实验中充分利用荧光的多种特性。[1-3]我们展示了一种新的实验安排,通过同时获取荧光恢复曲线(FRAP)、荧光寿命成像(FLIM)和荧光各向异性成像(fam)来测量细胞内动力学。我们使用这种设置来获得绿色荧光蛋白(GFP)标记蛋白在活细胞中的翻译和旋转扩散特性。该方法可以同时提取荧光寿命、旋转相关时间和扩散特性,从而避免因细胞运动而产生过度的光漂白或伪影。它还可以测量每种方法本身无法测量的现象,例如扩散的同型二聚体。
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引用次数: 0
Label free assessment of live cell quality with total internal reflection microscopy 用全内反射显微镜对活细胞质量进行无标签评估
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154840
M. Mather
Cellular feedstocks underpin many Regenerative Medicine (RM) therapies. Suitable strategies for the large-scale manufacture are therefore required to ensure high quality RM products are produced consistently at an economically acceptable price. Allied to this is the need for measurement tools capable of characterising cell quality and its dependence on key manufacture parameters in-process. Currently, cell populations are routinely monitored to assess quality using conventional biological analysis (e.g. cell surface markers, gene expression). This approach is destructive, not suitable for in-process measurements and renders time course experiments impossible. Alternatively non-destructive approaches that assess cell morphology can also used, with light microscopy techniques (e.g. bright field, phase contrast imaging) being the primary methods. Often these microscopy techniques are combined with pre-treatment of cells with exogenous labels such as fluorescent markers. This can provide functional information but has the disadvantage that such cell modifications are invasive and potentially toxic to the cells. Label free approaches are also used and whilst this enables non-invasive monitoring of live cells in culture, such microscopy techniques are currently non-quantitative with characterisation fully dependent on the skills and experience of the operator. Image contrast and resolution are also often lacking making morphological assessments unreliable. Additionally, more complex parameters such as dynamic cell behaviour and cell-substrate interactions are needed to provide the necessary mechanistic insight to characterise cellular processes and as parameters for effective process design and quality control tools. This presentation will address the above issues through the development of a total internal reflection microscope (TIRM) to enable the quantitative study of cellular processes and live cell quality at high resolution and without the use of labels. TIRM is a non-fluorescent imaging technique which is based on the principle that an object with refractive index (n3) will scatter an evanescent field created when a light beam undergoes total internal reflection at an interface between two media with different refractive indices, such as glass (n1) and air (n2), where n3>n2. The key design considerations with respect to development of a TIRM instrument are discussed. In addition the application of TIRM as an optical imaging tool to non-invasively monitor the quality of cells in culture at higher resolution than traditional light microscopy (e.g. bright field and phase contrast imaging) to enable validation of manufacturing procedures in-process will be discussed.
细胞原料支持许多再生医学(RM)疗法。因此,需要适合大规模生产的策略,以确保高质量的RM产品以经济上可接受的价格持续生产。与此相关的是需要能够表征电池质量及其对过程中关键制造参数的依赖的测量工具。目前,使用常规生物分析(如细胞表面标记、基因表达)对细胞群进行常规监测以评估质量。这种方法是破坏性的,不适合在过程中测量,使时间过程实验不可能。另外,也可以使用评估细胞形态的非破坏性方法,光学显微镜技术(例如,明场,相对比成像)是主要方法。通常,这些显微镜技术与外源标记(如荧光标记)的细胞预处理相结合。这可以提供功能信息,但缺点是这种细胞修饰是侵入性的,对细胞有潜在的毒性。也使用无标签方法,虽然这可以对培养中的活细胞进行无创监测,但这种显微镜技术目前是非定量的,其特征完全取决于操作人员的技能和经验。图像对比度和分辨率也经常缺乏,使形态学评估不可靠。此外,需要更复杂的参数,如动态细胞行为和细胞-基质相互作用,以提供必要的机制洞察力来表征细胞过程,并作为有效过程设计和质量控制工具的参数。本报告将通过全内反射显微镜(TIRM)的发展来解决上述问题,以实现高分辨率和不使用标签的细胞过程和活细胞质量的定量研究。TIRM是一种非荧光成像技术,其原理是,当光束在两种不同折射率介质(如玻璃(n1)和空气(n2)之间的界面上发生全反射时,折射率为(n3)的物体会散射出倏逝光场,其中n3>n2。讨论了有关TIRM仪器开发的关键设计考虑。此外,将讨论TIRM作为光学成像工具的应用,以比传统光学显微镜(例如,明场和相对比成像)更高的分辨率非侵入性地监测培养细胞的质量,从而验证生产过程中的程序。
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引用次数: 2
Proximity grating microscopy 近距离光栅显微术
Pub Date : 2011-12-01 DOI: 10.1109/FOI.2011.6154821
M. Somekh, F. Hu, C. Chuang, C. See
Structured illumination microscopy (SIM) using grating excitation can be used to extend the bandwidth of fluorescent microscopy by approximately a factor of 2 in the linear regime. If some of the fluorescent molecules are saturated even greater improvements in resolution are possible; this may, however, lead to high levels of photobleaching and phototoxicity. In this paper we present preliminary results that show a simple grating structure separated by a propagation region (see figure 1) can improve the resolution by a far greater factor than this offering the opportunity for resolution close to 50nm. Our present results are proof of concept results on relatively low numerical aperture systems. The potential for the higher lateral resolution relies on the fact that (i) the grating structure does not depend on the illumination optics and can thus be finer than possible with a grating formed by illumination through the lens and (ii) the propagation region can be made from a material with high refractive index is possible with immersion oils.
使用光栅激发的结构照明显微镜(SIM)可用于将荧光显微镜的带宽在线性范围内延长约2倍。如果一些荧光分子是饱和的,分辨率就有可能得到更大的提高;然而,这可能导致高水平的光漂白和光毒性。在本文中,我们提出的初步结果表明,通过传播区域(见图1)分隔的简单光栅结构可以以更大的因素提高分辨率,从而提供接近50nm的分辨率的机会。我们目前的结果是在相对小数值孔径系统上的概念验证结果。更高横向分辨率的潜力依赖于以下事实:(i)光栅结构不依赖于照明光学元件,因此可以比通过透镜照明形成的光栅更精细;(ii)传播区域可以由具有高折射率的材料制成,可以使用浸没油。
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引用次数: 0
期刊
2011 Functional Optical Imaging
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