自动对焦算法评估线虫脂滴图像的研究

自动对焦是实现线虫自动化筛选的一个重要步骤．在光学显微镜系统中,通过采集同一个视野下不同焦面的图像,再通过清晰度评价函数对这些图像进行运算,得到的最大值被认为是最佳对焦位置．在本研究中,对16种常用的自动对焦算法以及最近提出的一些算法进行了评估,通过评估找出最适合线虫脂滴图像的自动对焦算法,从而搭建一套线虫脂滴自动化筛选系统．同时就对焦精度、运算时间、抗噪声能力、对焦曲线等特征进行了分析评价,结果表明,大多数算法对线虫脂滴图像都有较好的表现,特别是绝对Tenengrad算法在对焦精度上有最好的表现,我们将优选该算法应用到线虫脂滴自动化筛选系统中．     

Comparison of autofocus methods for automated microscopy

Traditional autofocus methods were designed for microscopes driven by single processor computers. As computers are developed that exploit massive parallelism when acquiring and analyzing images, parallel cellular logic techniques became available to focus automatically. This paper introduces the reader to both cellular logic techniques for autofocus and a new spectral moment autofocus measure. It then compares these methods with more traditional autofocus methods. It is shown that traditional methods based on measurements of image power-give the best results when tested on one set of real images and two sets of synthetic images. The next best methods are the cellular logic and spectral moment techniques, while the worst are those based on the image probability density function or histogram.     

Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis

Microscopy images must be acquired at the optimal focal plane for the objects of interest in a scene. Although manual focusing is a standard task for a trained observer, automatic systems often fail to properly find the focal plane under different microscope imaging modalities such as bright field microscopy or phase contrast microscopy. This article assesses several autofocus algorithms applied in the study of fluorescence-labeled tuberculosis bacteria. The goal of this work was to find the optimal algorithm in order to build an automatic real-time system for diagnosing sputum smear samples, where both accuracy and computational time are important. We analyzed 13 focusing methods, ranging from well-known algorithms to the most recently proposed functions. We took into consideration criteria that are inherent to the autofocus function, such as accuracy, computational cost, and robustness to noise and to illumination changes. We also analyzed the additional benefit provided by preprocessing techniques based on morphological operators and image projection profiling.     

Robust autofocusing in microscopy

BACKGROUND: A critical step in automatic microscopy is focusing. This report describes a robust and fast autofocus approach useful for a wide range of microscopic modalities and preparations. METHODS: The focus curve is measured over the complete focal range, reducing the chance that the best focus position is determined by dust or optical artifacts. Convolution with the derivative of a Gaussian smoothing function reduces the effect of noise on the focus curve. The influence of mechanical tolerance is accounted for. RESULTS: The method is shown to be robust in fluorescence, bright-field and phase contrast microscopy, in fixed and living cells, as well as in fixed tissue. The algorithm was able to focus accurately within 2 or 3 s, even under extremely noisy and low contrast imaging conditions. CONCLUSIONS: The proposed method is generally applicable in light microscopy, whenever the image information content is sufficient. The reliability of the autofocus method allows for unattended operation on a large scale.     

Estimating nanoscale deformation in bone by X-ray diffraction imaging method

Knowledge of internal stress-strain in bone tissue is important for clinical diagnosis and remedies. The inorganic mineral phase of apatite crystals in bone composite, because of its crystalline nature, provides a reliable way of measurement through X-ray diffraction system. Use of two-dimensional detector, imaging plate (IP), is considered to expedite the process with much more information, hence, is widely applied in the study of organization, stress, strain, etc. for crystalline substance. The distortion of Debye rings in the image obtained by IP can be directly related to the deformation in lattice plane of the crystals. Since X-ray diffraction method involves measurement at nano-level, proper focus on the extraction of data and corresponding analysis is needed. In the current work, we considered weighted average value of intensity to locate radius vectors along azimuthal direction in the diffracted rings from the primary array of digital data in steps of pixels. The widely applied approaches for profile shift measurement--peak shift and full width at half maximum (FWHM) of a peak, and shift of center of gravity of profile--were compared with a new concept of segmental shift (SS) proposed previously by the authors. We observed reliable and effective outcomes with higher precision in the consideration of SS while using IP as a detector. Our approach in this work for intensity integration and radius vector positioning especially add precision in such applications.     

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope’s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.     

An Inexpensive Microscope Lamp for Critical Illumination

A microscope lamp with light of adjustable intensity, which will afford critical illumination of a quality satisfactory for the most exacting cytological work, can be constructed for about $7.50. The essential parts needed are: auto headlight bulb, radio power transformer, radio “potentiometer”, and simple plano-convex lens. A ground glass is not used with 4 and 2 mm. objectives, a solid source of light being obtained by placing the bulb so that one filament coil is directly behind the other. This light source is brought to a focus by the condensing lens at a field diaphragm and this image is projected by the microscope condenser into the plane of focus of the microscope.     

Dynamic light scattering microscopy. A novel optical technique to image submicroscopic motions. I: theory

The theoretical basis of an optical microscope technique to image dynamically scattered light fluctuation decay rates (dynamic light scattering microscopy) is developed. It is shown that relative motions between scattering centers even smaller than the optical resolution of the microscope are sufficient to produce significant phase variations resulting in interference intensity fluctuations in the image plane. The timescale and time dependence for the temporal autocorrelation function of these intensity fluctuations is derived. The spatial correlation distance, which reports the average distance between constructive and destructive interference in the image plane, is calculated and compared with the pixel size, and the distance dependence of the spatial correlation function is derived. The accompanying article in this issue describes an experimental implementation of dynamic light scattering microscopy.     

基于图像处理的树叶面积测量系统

以数字图像处理技术为基础,对数码相机拍摄的含有参照硬板和树叶的照片进行图像分析处理,提出了获取树叶的实际面积测量系统的一种独特实现方案。采用此方案开发出相应的应用软件,应用于树叶面积的测量计算,获得十分满意的结果。     

Low-Dose Micro-CT Imaging for Vascular Segmentation and Analysis Using Sparse-View Acquisitions

The aim of this study is to investigate whether reliable and accurate 3D geometrical models of the murine aortic arch can be constructed from sparse-view data in vivo micro-CT acquisitions. This would considerably reduce acquisition time and X-ray dose. In vivo contrast-enhanced micro-CT datasets were reconstructed using a conventional filtered back projection algorithm (FDK), the image space reconstruction algorithm (ISRA) and total variation regularized ISRA (ISRA-TV). The reconstructed images were then semi-automatically segmented. Segmentations of high- and low-dose protocols were compared and evaluated based on voxel classification, 3D model diameters and centerline differences. FDK reconstruction does not lead to accurate segmentation in the case of low-view acquisitions. ISRA manages accurate segmentation with 1024 or more projection views. ISRA-TV needs a minimum of 256 views. These results indicate that accurate vascular models can be obtained from micro-CT scans with 8 times less X-ray dose and acquisition time, as long as regularized iterative reconstruction is used.     

Pseudo‐real‐time retinal layer segmentation for high‐resolution adaptive optics optical coherence tomography

We present a pseudo‐real‐time retinal layer segmentation for high‐resolution Sensorless Adaptive Optics‐Optical Coherence Tomography (SAO‐OCT). Our pseudo‐real‐time segmentation method is based on Dijkstra's algorithm that uses the intensity of pixels and the vertical gradient of the image to find the minimum cost in a geometric graph formulation within a limited search region. It segments six retinal layer boundaries in an iterative process according to their order of prominence. The segmentation time is strongly correlated to the number of retinal layers to be segmented. Our program permits en face images to be extracted during data acquisition to guide the depth specific focus control and depth dependent aberration correction for high‐resolution SAO‐OCT systems. The average processing times for our entire pipeline for segmenting six layers in a retinal B‐scan of 496 × 400 and 240 × 400 pixels are around 25.60 and 13.76 ms, respectively. When reducing the number of layers segmented to only two layers, the time required for a 240 × 400 pixel image is 8.26 ms.     

Evaluation of automated threshold selection methods for accurately sizing microscopic fluorescent cells by image analysis.

The accurate measurement of bacterial and protistan cell biomass is necessary for understanding their population and trophic dynamics in nature. Direct measurement of fluorescently stained cells is often the method of choice. The tedium of making such measurements visually on the large numbers of cells required has prompted the use of automatic image analysis for this purpose. Accurate measurements by image analysis require an accurate, reliable method of segmenting the image, that is, distinguishing the brightly fluorescing cells from a dark background. This is commonly done by visually choosing a threshold intensity value which most closely coincides with the outline of the cells as perceived by the operator. Ideally, an automated method based on the cell image characteristics should be used. Since the optical nature of edges in images of light-emitting, microscopic fluorescent objects is different from that of images generated by transmitted or reflected light, it seemed that automatic segmentation of such images may require special considerations. We tested nine automated threshold selection methods using standard fluorescent microspheres ranging in size and fluorescence intensity and fluorochrome-stained samples of cells from cultures of cyanobacteria, flagellates, and ciliates. The methods included several variations based on the maximum intensity gradient of the sphere profile (first derivative), the minimum in the second derivative of the sphere profile, the minimum of the image histogram, and the midpoint intensity. Our results indicated that thresholds determined visually and by first-derivative methods tended to overestimate the threshold, causing an underestimation of microsphere size. The method based on the minimum of the second derivative of the profile yielded the most accurate area estimates for spheres of different sizes and brightnesses and for four of the five cell types tested. A simple model of the optical properties of fluorescing objects and the video acquisition system is described which explains how the second derivative best approximates the position of the edge.     

An image-interpolation technique for the computation of optic flow and egomotion

 A technique for measuring the motion of a rigid, textured plane in the frontoparallel plane is developed and tested on synthetic and real image sequences. The parameters of motion – translation in two dimensions, and rotation about a previously unspecified axis perpendicular to the plane – are computed by a single-stage, non-iterative process which interpolates the position of the moving image with respect to a set of reference images. The method can be extended to measure additional parameters of motion, such as expansion or shear. Advantages of the technique are that it does not require tracking of features, measurement of local image velocities or computation of high-order spatial or temporal derivatives of the image. The technique is robust to noise, and it offers a simple, novel way of tackling the ‘aperture’ problem. An application to the computation of robot egomotion is also described. Received: 3 September 1993/Accepted in revised form: 16 April 1994     

Assessing potential errors of MRI-based measurements of pulmonary blood flow using a detailed network flow model

MRI images of pulmonary blood flow using arterial spin labeling (ASL) measure the delivery of magnetically tagged blood to an image plane during one systolic ejection period. However, the method potentially suffers from two problems, each of which may depend on the imaging plane location: 1) the inversion plane is thicker than the imaging plane, resulting in a gap that blood must cross to be detected in the image; and 2) ASL includes signal contributions from tagged blood in conduit vessels (arterial and venous). By using an in silico model of the pulmonary circulation we found the gap reduced the ASL signal to 64-74% of that in the absence of a gap in the sagittal plane and 53-84% in the coronal. The contribution of the conduit vessels varied markedly as a function of image plane ranging from ～90% of the overall signal in image planes that encompass the central hilar vessels to <20% in peripheral image planes. A threshold cutoff removing voxels with intensities >35% of maximum reduced the conduit vessel contribution to the total ASL signal to ～20% on average; however, planes with large contributions from conduit vessels underestimate acinar flow due to a high proportion of in-plane flow, making ASL measurements of perfusion impractical. In other image planes, perfusion dominated the resulting ASL images with good agreement between ASL and acinar flow. Similarly, heterogeneity of the ASL signal as measured by relative dispersion is a reliable measure of heterogeneity of the acinar flow distribution in the same image planes.     

γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis

The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.     

动脉粥样硬化模型-大耳白家兔脑血管在体成像

动脉粥样硬化是中老年人的常见病,对人类的健康危害很大。应用激光扫描共聚焦显微镜,在不同刺激药物作用下对动脉粥样硬化大耳白家兔的脑血管形态进行了活体的实时动态观察。从脑血管的荧光图像和血管横截面的荧光强度分布可获取正常与异常状态血管内血流状态的相关形态学和流变学参数,从而提供对血管的阻塞状况以及形成机理进行研究的重要信息。     

RADIONUCLIDE COMPUTED TOMOGRAPHY: APPLICATIONS IN CARDIOVASCULAR DISEASE

Tomography is an imaging modality in which information in one plane is displayed in focus while information from overlying and underlying planes is either blurred or eliminated. As a result, there is an improvement in the contrast of structures in the plane of interest. Computerized axial tomography (CAT) is an imaging modality in which a digital computer mathematically reconstructs the image of a plane or section from data collected perpendicular to the axis of that plane. The axis of the plane and the patient's long axis usually coincide so that the reconstructed images reveal transverse sections. In recent years, two types of computed tomography in medical imaging have generated a great deal of interest. These are X-ray transmission and radio-nuclide emission computed tomography.     

Directional selection in temporally replicated studies is remarkably consistent

Temporal variation in selection is a fundamental determinant of evolutionary outcomes. A recent paper presented a synthetic analysis of temporal variation in selection in natural populations. The authors concluded that there is substantial variation in the strength and direction of selection over time, but acknowledged that sampling error would result in estimates of selection that were more variable than the true values. We reanalyze their dataset using techniques that account for the necessary effect of sampling error to inflate apparent levels of variation and show that directional selection is remarkably constant over time, both in magnitude and direction. Thus we cannot claim that the available data support the existence of substantial temporal heterogeneity in selection. Nonetheless, we conject that temporal variation in selection could be important, but that there are good reasons why it may not appear in the available data. These new analyses highlight the importance of applying techniques that estimate parameters of the distribution of selection, rather than parameters of the distribution of estimated selection (which will reflect both sampling error and "real" variation in selection); indeed, despite availability of methods for the former, focus on the latter has been common in synthetic reviews of the aspects of selection in nature, and can lead to serious misinterpretations.     

A New Method for the Analysis of Soft Tissues with Data Acquired under Field Conditions

Analyzing soft-tissue structures is particularly challenging due to the lack of homologous landmarks that can be reliably identified across time and specimens. This is particularly true when data are to be collected under field conditions. Here, we present a method that combines photogrammetric techniques and geometric morphometrics methods (GMM) to quantify soft tissues for their subsequent volumetric analysis. We combine previously developed methods for landmark data acquisition and processing with a custom program for volumetric computations. Photogrammetric methods are a particularly powerful tool for field studies as they allow for image acquisition with minimal equipment requirements and for the acquisition of the spatial coordinates of points (anatomical landmarks or others) from these images. For our method, a limited number of homologous landmarks, i.e., points that can be found on any specimen independent of space and time, and further distinctive points, which may vary over time, space and subject, are identified on two-dimensional photographs and their three-dimensional coordinates estimated using photogrammetric methods. The three-dimensional configurations are oriented by the spatial principal components (PCs) of the homologous points. Crucially, this last step orients the configuration such that x and y-information (PC1 and PC2 coordinates) constitute an anatomically-defined plane with the z-values (PC3 coordinate) in the direction of interest for volume computation. The z-coordinates are then used to estimate the volume of the tissue. We validate our method using a physical, geometric model of known dimensions and physical (wax) models designed to approximate perineal swellings in female macaques. To demonstrate the usefulness and potential of our method, we use it to estimate the volumes of Barbary macaque sexual swellings recorded in the field with video images. By analyzing both the artificial data and real monkey swellings, we validate our method''s accuracy and illustrate its potential for application in important areas of biological research.