Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index

Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive‐index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high‐end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive‐index mismatch for high‐aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high‐angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point‐scanning confocal microscope and showed that a nearly index‐matched, micron‐sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles.     

Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry

Confocal or multiphoton microscopes, which deliver optical sections and three‐dimensional (3D) images of thick specimens, are widely used in biology. These techniques, however, are sensitive to aberrations that may originate from the refractive index structure of the specimen itself. The aberrations cause reduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct for aberrations in confocal microscopes using adaptive optics. In order to define the design specifications for such adaptive optics systems, one has to know the amount of aberrations present for typical applications such as with biological samples. We have built a phase stepping interferometer microscope that directly measures the aberration of the wavefront. The modal content of the wavefront is extracted by employing Zernike mode decomposition. Results for typical biological specimens are presented. It was found for all samples investigated that higher order Zernike modes give only a small contribution to the overall aberration. Therefore, these higher order modes can be neglected in future adaptive optics sensing and correction schemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.     

基于几何光学的图像矫正法在圆管PIV实验中的应用研究

粒子图像速度技术被广泛用于流体流动测量,介质折射率差异使光在圆管壁面发生偏折,导致图像失真,直接影响速度测量精度。本文建立了光学折射的物理模型,得到圆形管道中物点和图像点之间的函数关系进而得到矫正后图像的像素坐标,使用双线性插值算法得到像素灰度值重建出矫正后的粒子图像,最后根据多重网格迭代算法计算管内速度场。分别对流体进行管内静态流体与管内层流速度场测量实验,对比了光学矫正箱法、线性矫正以及基于光学模型的畸变矫正方法误差。结果表明,本文提出的基于几何光学的图像矫正方法精度优于光学矫正箱法和线性矫正方法,并通过静态与流动实验充分验证了所建立几何光学模型的准确性和有效性。     

Simulation of specimen-induced aberrations for objects with spherical and cylindrical symmetry

Wavefront aberrations caused by the refractive index structure of the specimen are known to compromise signal intensity and three‐dimensional resolution in confocal and multiphoton microscopy. However, adaptive optics can measure and correct specimen‐induced aberrations. For the design of an adaptive optics system, information on the type and amount of the aberration is required. We have previously described an interferometric set‐up capable of measuring specimen‐induced aberrations and a method for the extraction of the Zernike mode content. In this paper we have modelled specimen‐induced aberrations caused by spherical and cylindrical objects using a ray tracing method. The Zernike mode content of the wavefronts was then extracted from the simulated wavefronts and compared with experimental results. Aberrations for a simple model of an oocyte cell consisting of two spherical regions and for a model of a well‐characterized optical fibre are calculated. This simple model gave Zernike mode data that are in good agreement with experimental results.     

Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method

Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z‐stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal‐to‐background ratio and image contrast. Here, we present comparative studies on a variety of biological samples.     

Application of optical velocity measurements including a novel calibration technique for micron-resolution to investigate the gas flow in a model experiment for crystal growth

An important step in the fabrication of many modern semiconductor materials and devices is the crystal growth process. These processes take place in high-temperature furnaces using inert gas and other atmospheres. The flow in the gas phase influences the transport of crystal components, dopants and impurities and hence has a significant impact on the grown crystals. In this work, we study the flow in a simplified model of a crystal growth furnace. This model is made of PMMA filled with a Diesel mixture as a model fluid to match the refractive index of PMMA and to allow for measurements in the complex geometry. The comparability to the flow in a real furnace is ensured by matching the Reynolds number. Two optical measurement methods, Particle Image Velocimetry (PIV) and the Laser Doppler Velocity Profile Sensor (LDV-PS) are used to investigate the global flow field as well as the small-scale flow structures. A calibration model is developed for the LDV-PS to reduce systematic measurement errors caused by the refractive index of the model fluid from up to 1% to less than 0,1%. The results obtained in this study improve the understanding of the gas flow behavior inside a crystal growth furnace and provide reference data for simulation. The first analysis shows a highly unsteady flow with unexpected flow direction along the crystal and melt surfaces. The near-surface flow patterns are of particular relevance in crystal growth because of their large influence on the local heat and mass transport during the crystallization process.     

高精度测量光学玻璃折射率的一种新方法

摘要: 准确测量光学玻璃的折射率已成为了保证光学系统成像质量非常重要的环节。本文介绍了光学玻璃折射率测试方法和国内外发展现状。提出了一种利用测量棱镜三个顶角所对应的三个最小偏向角的折射率测量新方法,该方法的特点是测量精度随被测光学玻璃的折射率增大而提高。在精度0.5²的测角仪上,折射率测量精度高于1x10-6。文中推导出了新方法的计算公式,并对精度做了理论分析。该方法由于测量精度高,有较好的应用前景。     

Sensitivity-enhanced single-mode fiber-tapered hollow core fiber-single-mode fiber Mach–Zehnder interferometer for refractive index measurements

In this paper, a sensitive-enhanced single-mode fiber—tapered hollow core fiber—single-mode fiber Mach–Zehnder interferometer is demonstrated for refractive index sensing. The sensitivity was improved by forming an up-taper at the two splicing joints and concave cone in hollow core fiber. The up-tapered regions served as a more effective mode splitter/combiner, and the tapered hollow core fiber was used to generate a stronger evanescent field to enhance the interaction of light with the analyte. According to the principles of interference between the cladding and fundamental modes, we performed refractive index measurements. The experiments indicated that the proposed sensor has a high refractive index sensitivity of 214.97?nm/RIU in the refractive index range of 1.333–1.379, with a minimum refractive index measurement resolution of 9.3?×?10^?5. In addition, the sensor had a low temperature response of 2.96?pm/°C in the range from 50 to 85°C and a low cross sensitivity of 1.377?×?10^?5 RIU/°C. The proposed sensor is attractive for its high refractive index sensitivity, easy fabrication, low cross sensitivity, and good mechanical strength, making it of potential value for refractive index measurements for chemical and biological sensing.     

Review of certain key issues in indirect measurements of the mass flow rate of solids in pneumatic conveying pipelines

On-line mass flow measurement of particulate solids in pneumatic conveying pipeline is a technically challenging area, where mass flow measurement presents a range of problems. These problems are not normally relevant to a single phase flow, but are always involved in gas–solids two-phase flows, like inhomogeneous distribution of solids over the pipe cross section, irregular velocity profiles, variations in particle size, moisture content, and deposition of fine particles on the inner wall of the pipeline. These variables may affect the response of a solids flow meter in ill-defined ways. All of these make the design and the calibration of solids’ mass flowmeter more difficult. Based on a review of non-invasive mass flow measurements of particulate solids, this paper summarizes and highlights several key issues, which often rely on structures of sensors or measurement methods, in indirect mass flow metering of pneumatically conveyed solids. They are: (i) spatial filtering effect; (ii) averaging effect; (iii) measurement resolution and sensitivity of array structures in tomography sensors.     

Continuously recording refractive index spectrograph for transparent and opaque insulators and semiconductors

An apparatus is described which enables the user to measure continuous refractive index spectra of opaque and transparent optically flat samples. The method of measurement is based on finding the Brewster's angle beta(B) of the sample. By modulating the angle of light incidence, the output signal of a lock-in amplifier, tuned to the modulation frequency, changes sign abruptly at beta(B). The lock-in signal is applied to a dc motor which drives a goniometer in that way that the sample is continually held at beta(B). Provided that the sample extinction coefficient k < or approximately 0.5, the accuracy of the refractive index measurements is better than 1%, or even 0.1% where k is approximately equal to 0.     

High sensitivity internal refractive index sensor based on a photonic crystal fiber long period grating

A high sensitivity method is reported for the measurement of the internal refractive index using a photonic crystal fiber long period grating. Long period gratings of different lengths were inscribed in photonic crystal fibers, and the air holes of the fiber had varying refractive indices. A side lobe appeared near the resonant dip through the analysis of the characteristics of transmission spectra showing variation in the refractive index. The resonant dip and its side lobe provided varying sensitivities to the internal refractive index values. The sensitivity of the side lobe was as high as 2343?nm/refractive index unit (RIU), which exceeded the value for the resonant dip (2047?nm/RIU) for refractive indices from 1.3333–1.3792. Due to the high resolution of 8.5?×?10^?6 RIU, this method offers promising applications for biological and chemical analysis in which high-precision refractive index measurements are required.     

Surface wave velocity measurement using reflections from laser scribed grooves

The measurement of ultrasonic surface wave (USW) velocity is difficult using the pulse overlap method (POM) in the presence of an externally applied stress. Coupling variations between the sample and the transducer cause nonuniform distortions of the two received pulses which makes the overlap condition difficult to determine over an extended stress range. The adaptation in this paper of a single transducer configuration to generate and detect the USW eliminates coupling variations between two or more transducers. In addition, two parallel, precisely positioned and contoured grooves are laser scribed on the sample surface to provide the necessary overlap echoes. Since both echoes are reflected only once by identical grooves and are equally affected by the distortions due to coupling variations, the overlap condition is relatively unaffected by the external stress and the accuracy of the USW velocity measurement is increased significantly.     

基于光纤激光频率分裂效应的折射率 / 厚度 双参量测量研究

折射率作为光学系统中应用最广泛的光学参数之一,对系统的光学性能具有极其重要的影响。 厚度与折射率所组成的 光学长度直接影响双折射器件在光学系统中的时延特性。 本文提出一种基于光纤激光频率分裂效应的折射率/ 厚度双参量测 量方法。 该系统通过对插入激光腔内的双折射器件进行旋转,利用频率分裂效应对不同角度的器件的双折射参数进行测量。 基于双折射器件中的折射率椭球,建立相位延迟、折射率、厚度和旋转角度之间的关系,通过拟合计算得到器件的折射率/ 厚度 参数。 实验结果表明,通过该系统对双折射元件的厚度测量误差为 210 nm,本征折射率进行测量误差为 10 -5 ,可广泛应用于红 外波段的双折射器件的本征折射率/ 厚度双参量测量。     

Modeling and verifying non-linearities in heterodyne displacement interferometry

The non-linearities in a heterodyne laser interferometer system occurring from the phase measurement system of the interferometer and from non-ideal polarization effects of the optics are modeled into one analytical expression which includes the initial polarization state of the laser source, the rotational alignment of the beam splitter along with different transmission coefficients for polarization states and the rotational misalignment of the receiving polarizer. The model is verified using a Babinet Soleil Compensator allowing a common path for both polarization states and thereby reducing the influence of the refractive index of air. The verification shows an agreement of the model with measurements with a standard deviation of 0.2 nm. With the use of the model it is confirmed that the mean of two polarizer receivers can reduce the effect of non-linearity. However, depending on the accuracy of the polarizer angles, a second-order non-linearity remains. Also the effect of rotational misalignment of the beam splitter can not be reduced in this way.     

压力信号干扰抑制的质量流量数据融合研究

质量流量测量精度受压力的影响,且随着压力的增大其测量精度变差。采用多个质量流量传感器进行多处测量,对质量流量测量数据进行自适应加权融合。在此基础上,为了消除压力对流量测量值的影响,采用BP神经网络进行压力干扰抑制的质量流量数据融合研究。研究结果表明,BP神经网络质量流量融合值的精度较自适应加权融合值的精度大大提高,且附加动量法获得的BP网络融合精度最高,自适应学习速率调整法次之,梯度下降法最差。     

热障涂层折射率与厚度的太赫兹无损检测

精确提取陶瓷层（Top coat,TC）与热生长氧化层（Thermally grown oxide,TGO）层在太赫兹频段的折射率是进行热障涂层（Thermal barrier coatings,TBCs）太赫兹无损检测研究的重要条件。由于对涂层样品只能采取反射式测量,所以首先比较了反射式与传统透射式测量条件下提取样品太赫兹光学参数及厚度的结果,随后利用反射式太赫兹时域脉冲成像系统提取等离子体喷涂的8YSZ热障涂层（TBCs）中TC层与TGO层的折射率,并依据所提取折射率进一步对TC层的厚度分布进行测量及成像。试验结果表明在材料中衰减较小的有效频段下反射式测量同样可以精确提取样品的折射率以及厚度,反射式测量TC层的平均折射率为5.23,TGO层的折射率为2.91,TGO的主要成分α-Al₂O₃的折射率为2.85。TBCs样品中TC层的平均厚度为257 μm,从TC层厚度的太赫兹图像中可观察到TC与粘结层（Bond coat,BC）界面的不均匀程度。反射式太赫兹无损检测可精确提取TBCs中TC与TGO的折射率和厚度,这对于TBCs中裂纹和气泡等缺陷的识别以及TGO生长太赫兹检测具有重要意义。     

基于洛伦兹模型的红外薄膜材料光学常数拟合及应用

在中长波红外区域,通常使用的镀膜材料都存在相当大的色散和一定的吸收。就目前的测试技术来说,确定这些材料的色散和吸收相当困难。试验基于洛伦兹谐振子模型对热蒸发制备的锗、硫化锌以及稀土氟化物薄膜的红外透射光谱进行拟合,得出这些材料在中长波红外区的光学常数。使用锗、硫化锌和稀土氟化物分别作为高折射率层(H)、中间折射率层(M)和低折射率层(L),设计并制备了从6.8μm~15μm的锗窗口宽带增透膜系,测量结果完全可以满足光机系统的要求。     

透明毛细管管壁折射率的无损测量

介绍了一种无损测量透明毛细管管壁折射率的方法。该方法利用平行光经过装有不同已知折射率标准液体的毛细管后会聚焦点位置不同的原理,通过CCD对会聚焦点位置成像后的图像进行判断,计算出毛细管外轮廓位置和焦距,进而计算出毛细管管壁的折射率,其测量精度可达0.005。分析了毛细管内外径对管壁折射率测量的影响,结果表明,管壁越厚,测量精度越高;同时对毛细管管壁折射率灵敏度和成像系统的景深值进行了计算和比较,结果显示,标准液体折射率越低,标定精度越高。该方法使用设备简单,操作方便,便于对图像观察和识别,实现了对毛细管管壁折射率的无损精确测量。     

表面等离子体共振理论仿真研究

为了掌握影响激发表面等离子体共振(SPR)现象的因素,文中以薄膜光学理论为基础,利用Winspall软件模拟研究Kretschman结构下,金膜厚度、待测介质折射率以及三棱镜类型对表面等离子体共振吸收峰位变化的影响,得出了最佳膜厚以及影响激发SPR现象的棱镜折射率和待测物质折射率随共振角度变化的规律,为SPR传感器的设...     

The effects of refractive index heterogeneity within kidney tissue on multiphoton fluorescence excitation microscopy

Although multiphoton fluorescence excitation microscopy has improved the depth at which useful fluorescence images can be collected in biological tissues, the reach of multiphoton fluorescence excitation microscopy is nonetheless limited by tissue scattering and spherical aberration. Scattering can be reduced in fixed samples by mounting in a medium whose refractive index closely matches that of the fixed material. Using optical 'clearing', the effects of refractive index heterogeneity on signal attenuation with depth are investigated. Quantitative measurements show that by mounting kidney tissue in a high refractive index medium, less than 50% of signal attenuates in 100 μm of depth.