Using biased image analysis for improving unbiased stereological number estimation – a pilot simulation study of the smooth fractionator

The smooth fractionator was introduced in 2002. The combination of a smoothing protocol with a computer‐aided stereology tool provides better precision and a lighter workload. This study uses simulation to compare fractionator sampling based on the smooth design, the commonly used systematic uniformly random sampling design and the ordinary simple random sampling design. The smooth protocol is performed using biased information from crude (but fully automatic) image analysis of the fields of view. The different design paradigms are compared using simulation in three different cell distributions with reference to sample size, noise and counting frame position. Regardless of clustering, sample size or noise, the fractionator based on a smooth design is more efficient than the fractionator based on a systematic uniform random design, which is more efficient than a fractionator based on simple random design. The fractionator based on a smooth design is up to four times more efficient than a simple random design.     

Quantification of microvessels in canine lymph nodes

Quantification of microvessels in tumors is mostly based on counts of vessel profiles in tumor hot spots. Drawbacks of this method include low reproducibility and large interobserver variance, mainly as a result of individual differences in sampling of image fields for analysis. Our aim was to test an unbiased method for quantifying microvessels in healthy and tumorous lymph nodes of dogs. The endothelium of blood vessels was detected in paraffin sections by a combination of immunohistochemistry (von Willebrand factor) and lectin histochemistry (wheat germ agglutinin) in comparison with detection of basal laminae by laminin immunohistochemistry or silver impregnation. Systematic uniform random sampling of 50 image fields was performed during photo-documentation. An unbiased counting frame (area 113,600 microm(2)) was applied to each micrograph. The total area sampled from each node was 5.68 mm(2). Vessel profiles were counted according to stereological counting rules. Inter- and intraobserver variabilities were tested. The application of systematic uniform random sampling was compared with the counting of vessel profiles in hot spots. The unbiased estimate of the number of vessel profiles per unit area ranged from 100.5 +/- 44.0/mm(2) to 442.6 +/- 102.5/mm(2) in contrast to 264 +/- 72.2/mm(2) to 771.0 +/- 108.2/mm(2) in hot spots. The advantage of using systematic uniform random sampling is its reproducibility, with reasonable interobserver and low intraobserver variance. This method also allows for the possibility of using archival material, because staining quality is not limiting as it is for image analysis, and artifacts can easily be excluded. However, this method is comparatively time-consuming.     

Sampling problems in an heterogeneous organ: Quantitation of relative and total volume of pancreatic islets by light microscopy

In stereological studies analysis of sampling variances is used for optimizing the sampling design. In organs with a heterogeneous distribution of the phase of interest the analysis of sampling variances can be undertaken only if the observed variance between sections is distributed into the fraction which is due to random variation and the fraction which is due to the heterogeneity. In the present example (pancreatic islet volume estimated by light microscopic point-counting) the density of islets showed a linear increase along the axis of the organ. By analysis of sampling variances it was calculated that the most efficient number of sections (cut perpendicular to the organ) was considerably lower when the isolated contribution from the random variation was considered. The total islet volume was obtained by the product of the fractional islet volume and the pancreatic weight. Analysis of sampling variances of the total islet volume was performed by including the variance contribution from the individual pancreatic weights to the variance of the group mean total islet volume. Due to a negative correlation between the fractional volume and organ weight the total islet volume in the group of animals was more precisely estimated than the fractional islet volume. The methods used for dealing with the heterogeneity of the organ and for estimating sampling variances of total structural quantities generalize to a large number of stereological studies in biology.     

Directional analysis of digitized planar sets by configuration counts

The oriented rose of normal directions of a random set Z can be used to quantify its anisotropy. A method for estimating this quantity from a digitization of Z in a sampling window, i.e. its pixel image, is presented. The image is analysed locally by considering pixel squares of size n × n. This allows us to count the number of different types of n × n configurations in the pixel image. We show that it suffices to restrict attention to the so‐called informative configurations. The number of informative configurations increases only polynomially in n. An algorithm to find these informative configurations is presented. Furthermore, estimators of the oriented rose based on counts of observed informative configurations are derived. The procedure is illustrated by a simulated example and an analysis of a microscopic image of steel.     

基于连续扫描方式的激光共焦扫描显微镜的研制

研发了一套基于连续扫描的激光共焦扫描显微镜(laser confocal scanning microscopy,LCSM)系统。该系统采用工作台连续运动方式实现扫描,提出了利用单次采集的数据滤除随机噪声的方法,避免了多帧取平均对成像速度造成的影响。实现连续扫描的关键在于解决工作台运动与数据采集的同步问题,利用采集卡有限采集模式,合理匹配工作台参数和采集参数,成功解决了这一问题。详细介绍了影响分辨率的因素,通过合理选取探测器针孔直径,取样间隔,确保了实现高分辨率的要求。系统利用Visual C#开发的控制平台,成功地对生物细胞进行了扫描成像。实验结果表明:基于连续扫描的LCSM具有较高的分辨率,生成的显微图像没有任何畸变,并且成像速度有了大幅度提高。     

High-throughput determination of grain size distributions by EBSD with low-discrepancy sampling

Because microstructure plays an important role in the mechanical properties of structural materials, developing the capability to quantify microstructures rapidly is important to enabling high-throughput screening of structural materials. Electron backscatter diffraction (EBSD) is a common method for studying microstructures and extracting information such as grain size distributions (GSDs), but is not particularly fast and thus could be a bottleneck in high-throughput systems. One approach to accelerating EBSD is to reduce the number of points that must be scanned. In this work, we describe an iterative method for reducing the number of scan points needed to measure GSDs using incremental low-discrepancy sampling, including on-the-fly grain size calculations and a convergence test for the resulting GSD based on the Kolmogorov–Smirnov test. We demonstrate this method on five real EBSD maps collected from magnesium AZ31B specimens and compare the effectiveness of sampling according to two different low discrepancy sequences, the Sobol and R₂ sequences, and random sampling. We find that R₂ sampling is able to produce GSDs that are statistically very similar to the GSDs of the full density grids using, on average, only 52% of the total scan points. For EBSD maps that contained monodisperse GSDs and over 1000 grains, R₂ sampling only required an average of 39% of the total EBSD points.     

A handheld support system to facilitate stereological measurements and mapping of branching structures

BranchSampler is a system for computer-assisted manual stereology written for handheld devices running Windows CE. The system has been designed specifically to streamline data collection and optimize sampling of tree-like branching structures, with particular aims of reducing user errors, saving time, and saving data in formats suited for further analysis in other software, for example, a spreadsheet. The system can be applied in a wide range of applications, from biomedical science to agriculture and horticulture. It can be applied for sampling nested generations of lung bronchioles and renal arterioles or for collection and optimizing sampling of crops for precision agriculture. Although the system has been designed specifically for sampling branching structures, it is sufficiently flexible to be used for other applications involving nested stereological designs. We describe the system specifications, software and Graphical User Interface development, functionality and application of the handheld system using four examples: (a) sampling monkey lung bronchioles for estimation of diameter and wall thickness (b) sampling rat kidney for estimating number of arteries and arterioles in a specific generation (c) mapping fruit (apple) tree yield in an orchard and (d) estimating the total leaf surface area of chrysanthemum plants in a greenhouse.     

Unbiased stereological estimation of the number and volume of nuclei and nuclear size variability in invasive ductal breast carcinomas

The application of design-based stereological methods for estimating nuclear features quantitatively in invasive ductal breast cancer is described. Nuclear number, size and size variability are explored in relation to the tumour grade and patient prognosis. The study includes an examination of the efficiency in estimating different nuclear volumes, and two different estimators of the nuclear size variability are contrasted. Forty-two invasive ductal breast carcinomas diagnosed and graded by two pathologists were used. Both 5-μm and 25-μm-thick sections were obtained from paraffin blocks for stereological study. More undifferentiated tumours show significantly larger nuclei than low-grade tumours. The estimates based on the disector method demonstrate a decrease in the number of tumour cell nuclei per unit volume of tissue from grades 1 to 2 and especially from grades 2 to 3. The univariate survival analysis shows a high prognostic value of the nuclear volume estimates. The study shows that an efficient sampling procedure was performed, particularly when estimating volume-weighted mean nuclear volume using the point-sampled intercepts method. This method is more efficient than estimation of the number-weighted mean nuclear volume using the selector method; however, the latter provides paired estimates of volume- and number-weighted mean nuclear volume, as well as an estimate of the coefficient of variation of nuclear volume in the number distribution of the same cells.     

Fractal compression and adaptive sampling: reducing the image acquisition time in scanning probe microscopy

In typical scanning probe microscope experiment a three‐dimensional image of a substrate is obtained. For a given scanning mechanism, the time needed to image an area depends mainly on the number of samples and the size of the image. The imaging speed is further compromised by drifts associated with the substrate and the piezoscanner. It is therefore desirable to improve the imaging speed with limited impact to the effective resolution of the resulting image. By utilizing an adaptive sampling scheme with fractal compression technique, we have demonstrated that the number of the required samples can be significantly reduced with minimal impact to the image quality. SCANNING 30: 463–473, 2008. © 2008 Wiley Periodicals, Inc.     

Estimation of the number of stellate cells in a liver with the smooth fractionator

To better evaluate the activation and proliferative response of hepatic stellate cells (HSC) in hepatic fibrosis, it is essential to have sound quantitative data in non‐pathological conditions. Our aim was to obtain the first precise and unbiased estimate of the total number of HSC in the adult rat, by combining the optical fractionator, in a smooth sampling design, with immunocytochemistry against glial fibrillary acidic protein. Moreover, we wanted to verify whether there was sufficiently relevant specimen inhomogeneity that could jeopardize the high expected estimate precision when using the smooth fractionator design for HSC. Finally, we wanted to address the question of what sampling scheme would be advisable a priori for future studies. Microscopical observations and quantitative data provided no evidence for inhomogeneity of tissue distribution of HSC. Under this scenario, we implemented a baseline sampling strategy estimating the number (N?) of HSC as 207E⁰⁶ (CV = 0.17). The coefficient of error [CE(N?)] was 0.04, as calculated by two formerly proposed approaches. The biological difference among animals contributed ? 95% to the observed variability, whereas methodological variance comprised the remaining 5%. We then carried out a half reduction of sampling effort, at the level of both sections and fields. In either occasion, the CE(N?) values were low (? 0.05) and the biological variance continued to be far more important than methodological variance. We concluded that our baseline sampling (counting 650–1000 cells/rat) would be appropriate to assess the lobular distribution and the N? of HSC. However, if the latter is the only parameter to be estimated, around half of our baseline sampling (counting 250–600 cells/rat) would still generate precise estimates [CE(N?) < 0.1], being in this case more efficient to reduce the number of sections than to reduce the sampled fields.     

Proper acquisition and handling of scanning electron microscopy images using a high-performance personal computer

The modern high-performance personal computer (PC) has very recently expanded the range of utilization of digital scanning electron microscopy (SEM) images, and the PC will be used increasingly with SEMs. However, the image quality of digital SEM images may be considerably influenced by scanning and digitization conditions. In particular, the effects of the aliasing error peculiar to digital data are often serious in the low-magnification acquisition (undersampling) of SEM images, and moreover even a high-magnification image (oversampling) is disturbed by the undersampled noise (a sort of aliasing error). Furthermore, the signal-to-noise ratio of a digitized SEM image is closely related to the performance of the analog-to-digital converter. To prevent a flood of low-quality digital images with artifacts by the aliasing and additional noise, we propose a method using very high-density sampling (scanning). In addition, we will discuss how to handle digital SEM images from the point of view of the sampling and quantization.     

基于DoG检测图像特征点的快速二进制描述子

针对SIFT描述子实时性差和传统二进制描述子对尺度、旋转和视角变化鲁棒性差的问题,本文通过优化采样模式和添加灰度差分不变量比较测试进行改进,提出了一种鲁棒性更高的二进制描述子。首先,设计了一种尺度关联、编号标记的采样模式;然后,旋转采样模式中各采样点到特定位置,确保描述子尺度、旋转不变性;接着,分析了采样点点对模式对描述子的影响,选择使用机器学习训练后的128对采样点对;最后,选择灰度值比较测试及梯度绝对值和比较测试构建二进制描述子。实验中采用DoG检测图像关键点,结果表明:本文提出的描述子在描述子构建和描述子匹配上比SIFT描述子分别快84%和67%;在有视角变化的图像匹配上,准确率比传统的二进制描述子高3%~5%,召回率平均要高30%以上。本文提出的特征点描述方法适用于时间要求高的图像匹配领域。     

低采样关联成像研究现状及发展

关联成像是一种基于光场二阶或高阶关联的主动间接成像技术,具有灵敏度高、抗干扰能力强等特点,在生物医学成像、遥感成像、海洋探测等领域有着重要的利用价值。然而,关联成像过程中对采样数的要求导致采样时间长、成像速度慢。如何在低采样的情况下获得高质量的图像重构依旧是关联成像在实际应用中需要解决的问题之一。详细阐述了关联成像的原理并介绍了近几年低采样关联成像的进展情况,如以深度学习为主的计算关联成像的理论及实验研究成果,并对关联成像的应用进行了总结和展望。     

Comparable morphometric data of pathological nerve obtained using the three-window sampling method and total fiber quantification

Morphometric analysis of nerve biopsy provides data of structural changes that are essential for early detection of peripheral neuropathy. Because of the laborious work associated with the total fiber quantification, various sampling methods have been introduced with controversial accuracy. Three-window sampling technique has been recently proposed to provide the accurate morphometric data of normal human sural nerve. However, its application in the diseased nerve has not been validated. This study, therefore, compared the morphometric data of nerve biopsies from 12 patients with various neuropathies obtained using this sampling method and those obtained by total fiber analysis. Total number, density, and diameter of myelinated fibers including myelin thickness and g ratio were analyzed. Intraclass correlation coefficients ranging from 0.95 to 0.99 indicate the high agreement between the data derived from the two methods in these parameters. This finding suggests the accuracy of the three-window sampling technique in the morphometric study of nerve with pathological alterations.     

An improved method for collecting and staining microorganisms for enumeration by fluorescence light microscopy

A rapid, robust method for the enumeration of total and viable microorganisms is described. A method using specific stains for viable and total cells and fluorescence light microscopy on membrane filters had been previously developed, but was sub-optimal in that some non-specific staining of the filters occurred and the filters were not flat enough for automatic image analysis methods to be employed, because not all cells in a field of view were in focus simultaneously. A new membrane filter has recently become available: the Anopore? membrane was described by the manufacturers as having a number of properties which would overcome these limitations. These include inorganic construction (giving resistance to solvents), high porosity (giving high flow rates), low surface adsorption (giving low background staining) and inherent hydrophilicity (simplifying wetting with aqueous solutions). Anopore membrane filters were found to produce very high contrast images of bacteria stained with ethidium bromide. Even with a relatively low power (magnification = 40) dry objective, these images could be easily thresholded for image analysis using only grey-level information. The methods developed here are considered to be a suitable basis for a fully automated procedure for the enumeration of total microbial populations.     

采样定理的拓展--一种新的非均匀采样规则

由于采样定量用来指导采样时段内信号的均匀采样（或重采样），但在实际中需要进行磋均匀采样，因此提出了一种新的非均匀采样规则，即让短时采样频率大于等于2倍的信号短时最大频率，以指导非均匀采样（或重采样）。经用两个仿真信号验证说明，非均匀采样的数据用样条插值恢复只有很小的数值误差，说明该规则是可行的，并将它与传统的非均匀采样定理做了比较，指出了它面向时变信号采样的特点。     

采样点分布对基于面形斜率径向基模型的自由曲面拟合精度的影响

鉴于自由曲面模型的面形拟合精度在自由曲面表征以及面形初始结构选取等研究中的重要性,本文针对基于面形斜率的高斯径向基表征模型,研究了不同的采样点分布类型对该模型面形拟合精度的影响。采用不同采样点分布拟合离轴二次曲面和带凸起的抛物面,结果表明采用均匀随机分布的采样点有利于实现高精度的面形拟合,且达到一定的拟合精度后,采样点的数目对拟合精度的影响有限。以离轴三反系统为设计实例,对比了由不同采样方式生成初始面形后系统的像质优化结果。结果显示,采用均匀随机型采样方式得到的初始面形进行系统优化,最终全视场平均调制传递函数(MTF)可以达到0.72以上,远高于由边缘集中采样方式生成初始面形后系统像质的优化结果,从而印证了理论研究结果。     

The estimation of measurement uncertainty of small circular features measured by coordinate measuring machines

This paper examines the measurement uncertainty of small circular features as a function of the sampling strategy; i.e., the number and distribution of measurement points. Specifically, we examine measuring a circular feature using a three-point sampling strategy in which the angular distance between the points varies from widely spaced, 120°, to closely grouped, a few degrees. Both theoretical and experimental results show that the measurement uncertainty is a strong function of the sampling strategy. The uncertainty is shown to vary by four orders of magnitude as a function of the angular distribution of the measurement points. A conceptual framework for theoretically estimating the measuring uncertainty is described, and good agreement with experiments is obtained when the measurements are consistent with the assumptions of the theoretical model. This paper is an expansion of a previous internal report1 with additional material on analog probes and probe lobing models.