Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics

Single-molecule fluorescence imaging is at the forefront of tools applied to study biomolecular dynamics both in vitro and in vivo. The ability of the single-molecule fluorescence microscope to conduct simultaneous multi-color excitation and detection is a key experimental feature that is under continuous development. In this paper, we describe in detail the design and the construction of a sophisticated and versatile multi-color excitation and emission fluorescence instrument for studying biomolecular dynamics at the single-molecule level. The setup is novel, economical and compact, where two inverted microscopes share a laser combiner module with six individual laser sources that extend from 400 to 640 nm. Nonetheless, each microscope can independently and in a flexible manner select the combinations, sequences, and intensities of the excitation wavelengths. This high flexibility is achieved by the replacement of conventional mechanical shutters with acousto-optic tunable filter (AOTF). The use of AOTF provides major advancement by controlling the intensities, duration, and selection of up to eight different wavelengths with microsecond alternation time in a transparent and easy manner for the end user. To our knowledge this is the first time AOTF is applied to wide-field total internal reflection fluorescence (TIRF) microscopy even though it has been commonly used in multi-wavelength confocal microscopy. The laser outputs from the combiner module are coupled to the microscopes by two sets of four single-mode optic fibers in order to allow for the optimization of the TIRF angle for each wavelength independently. The emission is split into two or four spectral channels to allow for the simultaneous detection of up to four different fluorophores of wide selection and using many possible excitation and photoactivation schemes. We demonstrate the performance of this new setup by conducting two-color alternating excitation single-molecule fluorescence resonance energy transfer (FRET) and a technically challenging four-color FRET experiments on doubly labeled duplex DNA and quadruple-labeled Holliday junction, respectively.     

基于声光调谐的扫频光源

提出了一种利用声光调谐方法进行滤波的扫频光源来提高它的输出稳定性。阐述了实现光源稳定输出的原理和方法,研究和分析了光源的相关参数。该系统采用声光调谐的方法代替机械滤波的方式。在一个环形腔内,使用半导体光放大器(SOA)作为增益介质,声光可调谐滤波器(AOTF)作为波长选择元件,利用声光调谐的原理对腔内的光进行选频滤波。在280mA的注入电流下,得到了1 294~1 368nm的扫频光源,其中心波长为1 328nm,半高全宽为51nm,扫频速度为3 731Hz,环形腔内直接输出的光功率为1.14mW。由于AOTF是电控制元件,波长的调谐不需要机械移动部件,故提高了系统的稳定性,输出光谱的重复性也很好。实验显示:通过这种方法获得的扫频光源输出稳定,基本满足扫频相干层析成像系统对扫频光源工艺参数的要求。     

近红外无创血糖测量的研究

本文分析了光在手掌组织中的传播特性 ,以及经皮近红外无创血糖浓度测量原理。基于AOTF (声光可调谐滤波器 )分光系统 ,构建了近红外经皮无创血糖检测系统 ,并利用该系统对 3名健康青年志愿者进行OGTT (口服葡萄糖耐量试验 )实验 ,得到较好的测量结果。三次实验中PLS (偏最小二乘 )校正集模型的RMSEP (预测均方根误差 )分别为 0 6 9mmol/L、 0 4 9mmol. /L、 0 5 4mmol/L。     

Background determination-based detection of scattered peaks

In many instances of signal and image processing, it is indispensable to precisely distinguish scattered peaks from a background, e.g., camera signals in microscopy. Here we addressed the detection of Gaussian signals in simulated line profiles (LP) comparable with e.g., fluorescence microscopy data. In a first step, we measured the applicability of histogram-based global background estimation. We find that the method is valid for typical scattered Gaussian signals if they are averagely separated by interpeak distances of 5.5 standard deviations. This enabled us to design global background determination-based peak detection (GBPD). GBPD was compared with two local background determination-based signal detection methods that had been designed for analysis of electrophysiological data and microscopy images, respectively. We were able to prove via receiver-operator characteristic (ROC) comparisons of signal-to-noise ratio (SNR), interpeak distance, and filtering behavior that, when applicable, GBPD brings advantages in knowledge needed a priori, performance at any SNR, controllability and spatial resolution.     

An efficient spectroscopic detection system for cathodoluminescence mode scanning electron microscopy (SEM)

A detection system for analytical cathodoluminescence (CL) mode scanning electron microscopy (SEM) is described. This incorporates a cold stage, an efficient light collector, a monochromator and a photomultiplier and used the photon counting technique. The efficiency of the component subsystems was optimized and calibrated, and the performance of alternative light collecting and monochromating equipment is compared. The operation of the photon counter is discussed. The digital output of the photon counter was fed into a multichannel scaler and thence to a computer. This was used to correct the observed count rate with the calibrated spectral variations in the performance of the detection system. Spectra obtained at both room temperature and liquid nitrogen temperacure and monochromatic and panchromatic SEM micrographs are given as examples illustrating the value of this technique. The factors governing the performance of the system are discussed. The forms of noise in the signal and in the detection system are described and the means for minimizing, avoiding or correcting for them are dealt with. Sources of spurious signals in the SEM are treated.     

基于柱矢量光束的纵向拉曼信号探测

提出了一种获得径向偏振光中纵向分量激发拉曼信号的方法,用于解决一般偏振拉曼信号无法检测分子取向垂直于样品的问题。首先使用聚焦后的角向偏振光和径向偏振光激发样品,然后用两种激发光产生的拉曼信号进行计算,得到由纯纵向光场激发的拉曼信号。使用Si(0 0 1)样品和Si(1 1 0)样品验证了该方法的可行性。这对于使用拉曼散射信号测量纵向取向的分子,以及纵向的分子振动模式具有一定意义。     

双重滤波扫频光源的研制

扫频光源是目前光学相干层析成像的关键部分,其光谱带宽和瞬时线宽分别影响着成像系统的轴向分辨率和成像深度。在单一滤波器中,这两者相互制约。针对这一情况,提出了一个利用两种滤波器组合优化的扫频光源系统。以双半导体光放大器并联作为增益介质,将声光可调滤波器(AOTF)和法布里-珀罗可调滤波器(FFP-TF)串联接入环形腔内进行双重滤波。其中AOTF的调谐范围和瞬时线宽均较宽,FFP-TF与之相反,经同步匹配设置后,两者协调工作,能够发挥各自的优势。通过搭建系统,获得了中心波长为1 316 nm的扫频激光输出,其光谱是1 235～1 380 nm,调谐范围是145 nm,瞬时线宽小于0.02 nm,扫频速度为1.35 kHz,输出光功率为0.48 mW。该扫频光源能够克服单一滤波器的固有缺陷,实现宽光谱带宽与窄瞬时线宽的有效统一,对成像综合性能的优化具有重要意义。     

超高分辨率CCD成像系统的设计

介绍了基于50 Mpixel超高分辨率全帧型CCD芯片KAF50100的成像系统设计方法.该系统采用幕帘式焦平面机械快门对CCD进行曝光控制,CCD输出图像信号在专用模拟前端(AFE)芯片AD9845B中进行处理和模数(A/D)转换后,经现场可编程门阵列(FPGA)缓存和排序,通过低压差分信号(LVDS)接口发送至上位机.系统中所有驱动时序和控制信号均由FPGA产生,上位机通过RS422总线对系统进行命令控制.针对KAF50100四路输出不均匀性问题提出了基于最小二乘法拟合的校正方法.实验验证表明,系统可在KAF50100的最大速度模式下工作,像素读出速度为4×18 MHz,最大帧速为1 frame/s,电路读出随机噪声为2.76@12bit,动态范围为63.4db.该成像系统设计方法可以充分发挥KAF50100的性能,并且具有良好的通用性和扩展性,可以广泛应用于超高分辨率CCD成像系统的设计中,如可见光水下探测、卫星遥感、天文观测等.     

Design of high performance QRS complex detector for wearable healthcare devices using biorthogonal spline wavelet transform

A high performance QRS complex detector applicable for wearable healthcare devices is proposed in the present work. Since, higher SNR results in better detection accuracy and lesser number of coefficients reduces the hardware resources as well as power dissipation during on chip implementation. Biorthogonal spline wavelet transform is chosen for the proposed detector as it has high signal to noise ratio (SNR) and uses only four coefficients for decomposition. In the proposed approach, a Biorthogonal wavelet filter bank with fourth level decomposition is first used to separate the different frequency components and then a fourth level wavelet filter bank is used to get the denoised electrocardiogram (ECG) signals. Wavelet filter bank outputs are multiplied and soft threshold method is applied to get the QRS complex peaks by the QRS complex peak detector block. Add and shift multiplier used in the earlier designs has been replaced by a Booth multiplier in our approach to achieve the higher performance. Booth multiplier and QRS complex peak detector blocks have been designed for low hardware complexity, high performance and accurate detection of the QRS complex peaks. Time interval between the consecutive QRS peaks is calculated using the R-R peak time calculator block and the heart rate (HR) by the HR calculator block. Heart Rate Variability (HRV) and arrhythmia are detected based on these heart rate calculations. Proposed design has been tested for its robustness on multiple datasets (namely, MIT-BIH arrhythmia, MIT-BIH noise stress test, and MIT-BIH atrial fibrillation databases). Sensitivity of 99.31%, positive predictivity of 99.19% and the Detection Error Rate (DER) of 1.49% shown by the proposed design makes it preferable for QRS complex detectors used in wearable healthcare devices.     

Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components

Using an optical system made from fused silica catalogue optical components, third‐order nonlinear microscopy has been enabled on conventional Ti:sapphire laser‐based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ～300 nm and in multimodal nonlinear optical imaging experiments using third‐order sum frequency generation and coherent anti‐Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ～300 nm to ～660 nm.     

Velocity sensing interferometer (VISAR) modification

The VISAR (velocity interferometer system for any reflector) has become a common tool used in experiments where high surface velocities must be measured. A modification that uses previously wasted interferometer light to more than double output signals and to cancel noise is described. Laser power is used more efficiently, VISAR performance in the presence of intense target self-light is improved, and only two data signals are required instead of the usual three or four. Effects of changing light intensity and fringe visibility are eliminated using a novel detection system with a simplified solution for velocity.     

Ultrafast photon counting applied to resonant scanning STED microscopy

To take full advantage of fast resonant scanning in super‐resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue‐to‐digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (～50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time‐gated continuous wave STED technology to the usage of resonant scanning with hardware‐based time‐gating. The assembled system provides superb signal‐to‐noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant‐scanning continuous wave STED microscopy with online time‐gated detection.     

GIS 局部放电光学传感器仿真设计与试验研究

在气体绝缘开关设备(GIS)的光学局部放电检测中,光学传感器是检测系统的核心部件。该文针对导光柱光学局部放电传感器的设计开展了仿真和实验研究。首先,基于TracePro仿真软件,对导光柱的前置微结构和长度进行设计,提出了一种用于GIS局部放电检测的导光柱光学传感器。随后,搭建GIS光学局部放电实验平台,研究了不同前置微结构和长度的导光柱的实际检测效果。仿真与实验结果均表明：前置微结构为凹锥,长度与罐体内壁平齐的导光柱传感效果较好。仿真与实验结果一致,证明了设计的可行性和仿真模型的正确性。最后,在该仿真模型中设置了多个探测面和探测点,观察并分析了各探测面的总光辐射度分布图和各探测点的光辐射度值。不同探测点接收的光辐射度值与部件遮挡以及直射光强弱有关。以放电源y-115为例,P1探测面上的光斑集中在150°～210°附近,P2探测面上的最优探测点主要位于在30°和330°附近。研究成果将为GIS光学局部放电传感器的研制提供参考依据。     

Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling

Electron microscopy (EM) is traditionally employed as a follow‐up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on‐section fluorescence labelling with on‐section immunogold labelling. In addition to antibody labelling of LAMP‐1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane‐associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane‐associated antigens.     

Use of independent component analysis to improve signal‐to‐noise ratio in multi‐probe fluorescence microscopy

In conventional multi‐probe fluorescence microscopy, narrow bandwidth filters on detectors are used to avoid bleed‐through artefacts between probes. The limited bandwidth reduces the signal‐to‐noise ratio of the detection, often severely compromising one or more channels. Herein, we describe a process of using independent component analysis to discriminate the position of different probes using only a dichroic mirror to differentiate the signals directed to the detectors. Independent component analysis was particularly effective in samples where the spatial overlap between the probes is minimal, a very common case in cellular microscopy. This imaging scheme collects nearly all of the emitted light, significantly improving the image signal‐to‐noise ratio. In this study, we focused on the detection of two fluorescence probes used in vivo, NAD(P)H and ANEPPS. The optimal dichroic mirror cutoff frequency was determined with simulations using the probes spectral emissions. A quality factor, defined as the cross‐channel contrast‐to‐noise ratio, was optimized to maximize signals while maintaining spatial discrimination between the probes after independent component analysis post‐processing. Simulations indicate that a ～3 fold increase in signal‐to‐noise ratio using the independent component analysis approach can be achieved over the conventional narrow‐band filtering approach without loss of spatial discrimination. We confirmed this predicted performance from experimental imaging of NAD(P)H and ANEPPS in mouse skeletal muscle, in vivo. For many multi‐probe studies, the increased sensitivity of this ‘full bandwidth’ approach will lead to improved image quality and/or reduced excitation power requirements.     

Microscopy of biological sample through advanced diffractive optics from visible to X-ray wavelength regime

The aim of this report is to demonstrate a unified version of microscopy through the use of advanced diffractive optics. The unified scheme derives from the technical possibility of realizing front wave engineering in a wide range of electromagnetic spectrum. The unified treatment is realized through the design and nanofabrication of phase diffractive elements (PDE) through which wave front beam shaping is obtained. In particular, we will show applications, by using biological samples, ranging from micromanipulation using optical tweezers to X-ray differential interference contrast (DIC) microscopy combined with X-ray fluorescence. We report some details on the design and physical implementation of diffractive elements that besides focusing also perform other optical functions: beam splitting, beam intensity, and phase redistribution or mode conversion. Laser beam splitting is used for multiple trapping and independent manipulation of micro-beads surrounding a cell as an array of tweezers and for arraying and sorting microscopic size biological samples. Another application is the Gauss to Laguerre-Gauss mode conversion, which allows for trapping and transfering orbital angular momentum of light to micro-particles immersed in a fluid. These experiments are performed in an inverted optical microscope coupled with an infrared laser beam and a spatial light modulator for diffractive optics implementation. High-resolution optics, fabricated by means of e-beam lithography, are demonstrated to control the intensity and the phase of the sheared beams in x-ray DIC microscopy. DIC experiments with phase objects reveal a dramatic increase in image contrast compared to bright-field x-ray microscopy. Besides the topographic information, fluorescence allows detection of certain chemical elements (Cl, P, Sc, K) in the same setup, by changing the photon energy of the x-ray beam.     

Using c-Fos/c-Jun as hetero-dimer interaction model to optimize donor to acceptor concentration ratio range for three-filter fluorescence resonance energy transfer (FRET) measurement

Sensitized emission FRET detection method based on three-filter fluorescence microscopy is widely used and more suitable for live cell FRET imaging and dynamic protein-protein interaction analysis. But when it is applied to detect two proteins interaction in living cells, this intensity-based detection method is complicated by many experimental factors such as spectral crosstalk and spectral bleed-through and variable donor to acceptor concentration ratio. There are several FRET algorithms developed recently to correct those factors in order to quantitatively gauge and compare FRET signals between different experimental groups. But the algorithms are often difficult to choose when they are applied to certain experiments. In this research, we use c-Fos/c-Jun as a simple hetero-dimer interaction model to quantitatively detect and compare the FRET signals based on the following widely used sensitized emission FRET algorithms: N(FRET) , FRET(N) , FR, FRET(R) , E(app) and E(EFF) . We optimized the donor to acceptor concentration ratio range for the above FRET algorithms and facilitate their use in accurate FRET signal determination based on the three-filter FRET microscopy.     

基于AOTF技术的光纤损耗光谱检测系统设计

提出了一种新型的基于声光可调滤光器(AOTF)的便携式光纤损耗光谱检测系统。建立了光纤损耗光谱检测的数学模型和检测系统总体结构。该系统利用声光可调滤光器的电可调谐特性,采用DSP进行智能化控制,实现对光纤中信号光的快速光谱扫描,测量出被测光纤对不同波长的信号光的衰减特性。最后给出了实验结果和讨论。     

A multi-detector, digitizer based neutron depth profiling device for characterizing thin film materials

Neutron depth profiling (NDP) is a mature, nondestructive technique used to characterize the concentration of certain light isotopes in a material as a function of depth by measuring the residual energy of charged particles in neutron induced reactions. Historically, NDP has been performed using a single detector, resulting in low intrinsic detection efficiency, and limiting the technique largely to high flux research reactors. In this work, we describe a new NDP instrument design with higher detection efficiency by way of spectrum summing across multiple detectors. Such a design is capable of acquiring a statistically significant charged particle spectrum at facilities limited in neutron flux and operation time.     

弹光和电光级联的组合相位调制型椭偏测量术

利用弹光调制器的偏振调制优势,提出了将弹光调制器和电光调制器级联的组合相位调制型椭偏测量术。该技术采用单光路实现信号探测,通过切换电光调制器的两个工作状态,并结合数字锁相信号处理技术来完成样品反射光的p分量和s分量的幅值比Ψ和相位差Δ的全范围高精度测量。对提出的新型椭偏测量原理进行了分析,搭建了相应的实验系统。完成了弹光调制器的调制电压峰峰值和相位调制幅值关系的定标,定标结果优于99.05%,然后还采用系统初始偏移值的方法对实验系统进行了校准。最后,运用该系统对石英玻璃反射样品进行了实验分析,得到的Ψ和Δ的测量精度分别优于0.08°和0.81°。实验结果显示系统校准有效地消除了电光调制器和弹光调制器的剩余双折射引入的测量误差;数据采集和处理时间均在ms量级。提出的测量技术具有宽光谱测量、工作稳定、重复度高、测量速率快、成本相对较低和系统便于工业自动化集成的潜在优势。