Detection of Single Quantum Dots in Model Systems with Sheet Illumination Microscopy

Single molecule detection and tracking provides at times the only possible method to observe the interactions of low numbers of biomolecules, inlcuding DNA, receptors and signal mediating proteins in living systems. However, most existing imaging methods do not enable both high sensitivity and non-invasive imaging of large specimens. In this study we report a new setup for selective plane illumination microscopy (SPIM), which enables fast imaging and single molecule tracking with the resolution of confocal microscopy and the optical penetration beyond 300 μm. We detect and report our instrumental figures of merit, control values of fluorescence properties of single nano crystals in comparison to both standard widefield configurations, and also values of nanocrystals in multicellular “fruiting bodies” of Dictyostelium, an excellent control as a model developmental system. In the Dictyostelium , we also report some of our first tracking of single nanocrystals with SPIM. The new SPIM setup represents a new technique, which enables fast single molecule imaging and tracking in living systems.     

Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM)

Multidirectional selective plane illumination microscopy (mSPIM) reduces absorption and scattering artifacts and provides an evenly illuminated focal plane. mSPIM solves two common problems in light-sheet-based imaging techniques: The shadowing in the excitation path due to absorption in the specimen is eliminated by pivoting the light sheet; the spread of the light sheet by scattering in the sample is compensated by illuminating the sample consecutively from opposing directions. The resulting two images are computationally fused yielding a superior image. The effective light sheet is thinner, and the axial resolution is increased by square root 2 over single-directional SPIM. The multidirectional illumination proves essential in biological specimens such as millimeter-sized embryos. The performance of mSPIM is demonstrated by the imaging of live zebrafish embryos.     

基于CCD虚拟针孔探测的双轴共焦显微技术

双轴共焦显微技术具有独特的非共轴结构,与传统单轴共焦显微技术相比可利用较低数值孔径物镜实现较高的轴向分辨力,且具有工作距离大、信噪比高等优势。对基于CCD虚拟针孔(VPH)探测的双轴共焦显微成像系统的空间分辨特性进行了理论分析,并构建了相应的实验系统,对其轴向响应进行了实验验证。实验中照明物镜NAi=0.117,采集物镜NAc=0.106,θ=45°,得出系统轴向半高宽(FWHM)为2.63μm,比同等参数(NA=0.117)下单轴共焦显微系统的轴向FWHM高出约20倍。     

激光分光瞳共焦显微镜系统研制

为了满足生物类等样品对大工作距和高分辨率共焦显微镜的需求,将分光瞳技术与激光共焦显微技术结合应用到成像系统上。阐述了激光分光瞳共焦显微成像原理,首次成功搭建了相应的显微镜成像测量系统。理论分析和实验表明:分光瞳共焦显微技术独特的非共轴结构使系统的轴向分辨力是相同数值孔径物镜单轴系统的3倍以上,对理论高度为100nm的台阶样品进行成像测试,得到的样品三维形貌,成像质量良好。     

Evaluation of Sensitivity of Fluorescence-Based Asbestos Detection by Correlative Microscopy

Fluorescence microscopy (FM) has recently been applied to the detection of airborne asbestos fibers that can cause asbestosis, mesothelioma and lung cancer. In our previous studies, we discovered that the E. coli protein DksA specifically binds to the most commonly used type of asbestos, chrysotile. We also demonstrated that fluorescent-labeled DksA enabled far more specific and sensitive detection of airborne asbestos fibers than conventional phase contrast microscopy (PCM). However, the actual diameter of the thinnest asbestos fibers visualized under the FM platform was unclear, as their dimensions were below the resolution of optical microscopy. Here, we used correlative microscopy (scanning electron microscopy [SEM] in combination with FM) to measure the actual diameters of asbestos fibers visualized under the FM platform with fluorescent-labeled DksA as a probe. Our analysis revealed that FM offers sufficient sensitivity to detect chrysotile fibrils as thin as 30–35 nm. We therefore conclude that as an analytical method, FM has the potential to detect all countable asbestos fibers in air samples, thus approaching the sensitivity of SEM. By visualizing thin asbestos fibers at approximately tenfold lower magnifications, FM enables markedly more rapid counting of fibers than SEM. Thus, fluorescence microscopy represents an advanced analytical tool for asbestos detection and monitoring.     

Morphologies and Mechanical Properties of High-Density Polyethylene Induced by the Addition of Small Amounts of Both Low- and High-Molecular-Weight Polyolefin under Shear Stress Applied by Dynamic Packing Injection Molding

This paper focuses on the mechanical properties and crystal morphology of a self-reinforced high-density polyethylene 5000S (HDPE 5000S) by simultaneously blending with 9 wt% high-molecular-weight polyethylene (HMWPE) and 9 wt% low-molecular-weight polyethylene (LMWPE) (A9) under the shear stress field which was engendered by a self-made dynamic packing injection molding (DPIM) machine. The results of mechanical properties, differential scanning calorimetry, and scanning electron microscopy characterization were as follows: (1) The tensile strength of the dynamic samples increased to 112.1 MPa, 4.85 times as much as that of static packing injection molding (SPIM) samples (23.1 MPa), as a result of realizing polyethylene's self-enhancement; (2) Shish-kebab structure was found in the dynamic samples; (3) The crystallinity of the DPIM A9 sample reached 68.6%, on increase by 18.7% compared with that of the SPIM sample. The formation of the shish-kebab structure and enhancement of mechanical properties are explained.     

微分干涉差共焦显微膜层微结构缺陷探测系统

多层膜极紫外光刻掩模"白板"缺陷是制约下一代光刻技术发展的瓶颈之一,为提高对掩模"白板"上的膜层微结构缺陷的分辨能力,提出了一种微分干涉差共焦显微探测系统方案。基于标量衍射理论,计算了系统横向和轴向分辨率。利用MATLAB建模仿真,在数值孔径为0.65、工作波长为405 nm时,分析比较了微分干涉差共焦显微系统、传统显微系统和共焦显微系统的分辨率。结果表明微分干涉差共焦显微系统具有230 nm的横向分辨率和25 nm轴向台阶高度差的分辨能力(对应划痕等缺陷形式)。此外,仿真和分析了实际应用中探测器尺寸、样品轴向偏移等的影响,模拟分析了膜层微结构缺陷的探测,结果表明本系统可以探测200 nm宽、10 nm高的微结构缺陷,较另外两种系统有更好的探测能力。     

Molecular vibration imaging in the fingerprint region by use of coherent anti-Stokes Raman scattering microscopy with a collinear configuration

We have developed a new coherent anti-Stokes Raman scattering (CARS) microscopy system with a collinear configuration for use in the fingerprint region. The system consists of a picosecond laser system and a transmission-type laser scanning microscope without a pinhole in front of the detector. The observable Raman-shift region is 900-1750 cm(-1), the spectral resolution is 30cm(-1), and the spatial resolution is smaller than 1 mum in the lateral direction and 3.2 mum in the depth direction, with objectives with a numerical aperture of 0.65. CARS spectra and images of polystyrene beads are demonstrated, and CARS imaging of a viable yeast cell is attempted.     

A lateral super-resolution differential confocal technology with phase-only pupil filter

In order to achieve a higher lateral resolution required for ultraprecision measurement of microstructural workpieces, phase-only pupil filtering differential confocal microscopy (PFDCM), a new approach is proposed based on the differential confocal microscopy (DCM), which uses a three-zone phase-only pupil filter with lateral super-resolution capability obtained through optimized design to change the distribution of DCM three-dimensional point spread function, so that the DCM lateral resolution is therefore significantly improved while its axial resolution is slightly improved. Preliminary experimental comparison and analyses indicate that, the lateral and axial resolutions of PFDCM are better than 0.2 μm and 2 nm, respectively, when wavelength of incidence laser beam , numerical aperture of measuring lens NA=0.85, and lateral spot size with a three-zone phase-only pupil filter G_T=0.65. It is therefore concluded that PFDCM is a new approach to further improvement of lateral resolution in laser probe measurement systems.     

Selective plane illumination optical and optoacoustic microscopy for postembryonic imaging

Intravital imaging of large specimens is intrinsically challenging for postembryonic studies. Selective plane illumination microscopy (SPIM) has been introduced to volumetrically visualize organisms used in developmental biology and experimental genetics. Ideally suited for imaging transparent samples, SPIM can offer high frame rate imaging with optical microscopy resolutions and low phototoxicity. However, its performance quickly deteriorates when applied to opaque tissues. To overcome this limitation, SPIM optics were merged with optical and optoacoustic (photoacoustic) readouts. The performance of this hybrid imaging system was characterized using various phantoms and by imaging a highly scattering ex vivo juvenile zebrafish. The results revealed the system's enhanced capability over that of conventional SPIM for high‐resolution imaging over extended depths of scattering content. The approach described here may enable future visualization of organisms throughout their entire development, encompassing regimes in which the tissue may become opaque.

     

Predicting phase shift effects for vibrating fluid-conveying pipes due to Coriolis forces and fluid pulsation

Knowing the influence of fluid flow perturbations on the dynamic behavior of fluid-conveying pipes is of relevance, e.g., when exploiting flow-induced oscillations of pipes to determine the fluids mass flow or density, as done with Coriolis flow meters (CFM). This could be used in the attempts to improve accuracy, precision, and robustness of CFMs. A simple mathematical model of a fluid-conveying pipe is formulated and the effect of pulsating fluid flow is analyzed using a multiple time scaling perturbation analysis. The results are simple analytical predictions for the transverse pipe displacement and approximate axial shift in vibration phase. The analytical predictions are tested against pure numerical solution using representative examples, showing good agreement. Fluid pulsations are predicted not to influence CFM accuracy, since proper signal filtering is seen to allow the determination of the correct mean phase shift. Large amplitude motions, which could influence CFM robustness, do not appear to be induced by the investigated fluid pulsation. Pulsating fluid of the combination resonance type could, however, influence CFMs robustness, if induced pipe motions go unnoticed and uncontrolled during CFM operation by feedback control. The analytical predictions offer an immediate insight into how fluid pulsation affects phase shift, which is a quantity measured by CFMs to estimate the mass flow, and lead to hypotheses for more complex geometries, i.e. industrial CFMs. The validity of these hypotheses is suggested to be tested using laboratory experiments, or detailed computational models taking fluid-structure interaction into account.     

Thermal Hall conductivity with sign change in the Heisenberg–Kitaev kagome magnet

The Heisenberg-Kitaev(HK) model on various lattices has attracted a lot of attention because it may lead to exotic states such as quantum spin liquid and topological orders.The rare-earth-based kagome lattice(KL) compounds Mg₂RE₃Sb₃O₁₄(RE=Gd,Er) and(RE=Nd) have q=0,120° order and canted ferromagnetic(CFM) order,respectively.Interestingly,the HK model on the KL has the same ground state long-range orders.In the theoretical phase diagram,the CFM phase re...     

具有高空间分辨力的整形环形光式差动共焦测量法

提出一种新的具有高空间分辨力的整形环形光式差动共焦测量方法。该方法通过整形环形光式共焦测量法和锐化爱里斑主瓣，改善系统横向分辨力；通过差动共焦测量法改善系统的轴向分辨力，最终达到提高系统空间分辨能力的目的。理论分析和实验表明：整形环形光内孔归一化半径ε越大，横向分辨力改善越明显，量程扩展范围越宽；当入射光波长λ=632.8nm，物镜数值孔径取NA=0.85，ε=0.5时，该系统的横向分辨力优于0.2μm，轴向分辨力优于2nm。该方法为光触针测量系统空间分辨力的提高提供了1种新的方法，可广泛应用于超精密三维微细结构工件的超精密测量。     

Exchange bias and domain evolution at 10 nm scales

For a fixed 2 μm×2 μm area of a Co/Pt-CoO perpendicular exchange bias system we image the ferromagnetic (FM) domains for various applied fields with 10-nm resolution by magnetic force microscopy (MFM). Using quantitative MFM we measure the local areal density of pinned uncompensated spins (pinUCS) in the antiferromagnetic (AFM) CoO layer and correlate the FM domain structure with the UCS density. Larger applied fields drive the receding domains to areas of proportionally higher pinUCS aligned antiparallel to FM moments. The data confirm that the evolution of the FM domains is determined by the pinUCS in the AFM layer, and also present examples of frustration in the system.     

小动物正电子断层成像仪性能测试

设计了一系列实验对Concorde MicroPET Rodent R4小动物正电子断层成像系统(该系统是Concorde Microsystems公司开发的小动物专用PET扫描仪，该扫描仪具有32层探测器环，动物入口孔径为120mm，横向视野为100mm，轴向深度为78mm，标称的视野中央处的空间分辨率可达1.8mm以下)的空间分辨率、灵敏度、散射率等性能参数进行了测试，测试结果显示该扫描仪视野中心的横向空间分辨率为1.9mm, 轴向空间分辨率为1.88mm, 在能窗设定为250—750keV时系统的绝对灵敏度为39.88 cps/kBq, 同样能窗下二维重建散射分数为50.6%,三维重建散射分数为32.3%. 关键词： 正电子断层成像仪 性能测试     

Axial super-resolution by mirror-reflected stimulated emission depletion microscopy

In stimulated emission depletion (STED) microscopy, the lateral resolution is in the range of tens of nanometers depending on the sample and the instrument. The axial resolution, however, is in standard systems limited by diffraction to about 500 nm. We present an approach to three-dimensional diffraction-unlimited resolution by observing the sample at two optical angles. The system is realized by using an atomic force microscope (AFM) chip as a microreflector to deflect the STED beams near the region-of-interest (ROI), thus allowing observations at an angle ∠. Consequently, the superior lateral resolution can be utilized to resolve details in the axial direction of the main optical axis of the microscope. Here, fluorescent nanoparticles 90 nm apart and biological structures 80 nm apart along axial direction were distinguished by utilizing an off-the-shelf, commercial STED microscope, coupled with an AFM and an AFM chip micro-reflector.     

Enhanced background rejection in thick tissue using focal modulation microscopy with quadrant apertures

Image formation in focal modulation microscopy using quadrant apertures (QFMM) is presented. The spatial resolution is discussed: compared with confocal microscopy, QFMM can simultaneously enhance the axial and transverse resolution. In contrast to focal modulation microscopy using D-shaped apertures (DFMM), QFMM can maintain x-y symmetry of spatial resolution, thus causing less confusion in imaging interpretation. The capability of background rejection is investigated, showing QFMM has the potential to increase imaging penetration depth. The signal level is also analyzed, indicating that the preferred detector pinhole radius is greater than for DFMM, resulting in a higher signal level.     

内窥式散斑类共聚焦系统层析能力分析

本文研究了内窥式散斑类共聚焦系统(Endoscope-based speckle quasi-confocal system,EBSQCS)的层析能力。从散斑类共聚焦显微镜(Speckle quasi-confocal microscope,SQCM)成像原理出发,详细分析了内窥镜光学结构对散斑类共聚焦显微镜散斑场波动的影响规律,推导了内窥镜光学结构与内窥式散斑类共聚焦系统轴向分辨力的关系。实验测得了基于光纤束的内窥式散斑类共聚焦系统的轴向分辨力曲线。选用放大倍率4倍,光纤直径5μm的内窥镜系统的内窥式散斑类共聚焦系统轴向分辨力曲线的全峰半高是散斑类共聚焦显微镜的2.3倍,与理论计算值相符。实验结果表明内窥式散斑类共聚焦系统具有很好的轴向层析能力。     

用于高数值孔径物镜的可调光瞳滤波器

根据矢量衍射理论，提出了一种用于提高高数值孔径显微物镜纵向分辨率的可调二元环形光瞳滤波器，通过改变滤波器的环半径获得不同的纵向分辨率，并利用共焦显微术点扩散函数的乘积性大大地降低了旁瓣相对主瓣的比值。模拟结果表明，这种新型滤波器不仅可以提高高数值孔径物镜的纵向分辨率，同时基本上不影响横向分辨率。     

改善共焦系统轴向分辨率的位相型光瞳滤波器

利用约束全局优化算法———CGO算法,设计了两种用于共焦系统的三区位相型光瞳滤波器.第一种滤波器在不改变系统的横向分辨率的同时,可以大幅度地提高轴向分辨率,提高了系统的层析能力.第二种滤波器在提高系统轴向分辨率的同时,又能提高其横向分辨率,适用于系统的三维成像. 关键词： CGO算法 光瞳滤波器 超分辨