Intercomparison of automatic fission track counting systems

The methods of counting fission tracks by two image analyzing computers and a spark counting system are compared with a standard microscopy technique. In this comparison, for automatic fission track counting, the two commercially available image analyzer instruments, the Quantimet 720 and the Leitz-Texture Analyzing System (TAS) and a specially designed spark counter, the UTASC system, have been used. The results showed that the relative counting efficiencies of the Quantimet 720, the Leitz-TAS and the UTASC system are 0.974±0.010, 0.988±0.013 and 0.955±0.010, respectively     

Modeling the clay minerals–enzyme binding by fusion fluorescent proteins and under atomic force microscope

In the present study, binding of cellulase protein to different clay minerals were tested using fluorescent–protein complex and microscopic techniques. Cellulase gene (Cel5H) was cloned into three fluorescent vectors and expressed as fusion enzymes. Binding of Cel5H–mineral particles was confirmed by confocal microscopy, and enzyme assay. Among the Cel5H–fusion enzymes, green–fusion enzyme showed higher intensity compared with other red and yellow fusion–proteins. Intensity of fusion–proteins was dependent on the pH of the medium. Confocal microscopy revealed binding of the all three fusion proteins with different clay minerals. However, montmorillonite displayed higher binding capacity than kaolinite clay. Likewise, atomic force microscopy (AFM) image profile analysis showed proteins appeared globular molecules in free‐state on mica surface with an average cross sectional diameter of 110 ± 2 nm and rough surface of montmorillonite made protein appear flattened due to structural alteration. Even surface of kaolinite also exerted some strain on protein molecular conformation after binding to surface. Our results provide further evidence for 3D visualization of enzyme–soil complex and encourage furthering study of the force involved interactions. Therefore, our results indicate that binding of proteins to clay minerals was external and provides a molecular method to observe the interaction of clay minerals–enzyme complex.     

3D restoration with multiple images acquired by a modified conventional microscope

A problem in high magnification microscopy is the blurring in the imaging of an object. In this article, we demonstrate a restoration technique that simultaneously makes use of the confocal image and the wide-field image. These images can be acquired by a modified conventional microscope. In front of the light-source, there is an array of pinholes. There are no pinholes at the detection plane. Instead, one or more pixels from the CCD camera are used, where the pinholes would have been. Using all pixels gives the wide-field image, but using a selected subset can give a confocal image. The array is used to speed up the process of acquiring the image. Note that the speed of acquisition is proportional to the number of pinholes. We show that the restoration from the two images can lead to a better result than using only one of the images. If this is the case, we show that a distance of 5 times the diameter of the pinholes can give the same results as a distance of 20 times after deconvolution. This offers an increase in acquisition time of a factor 16.     

The use of single image random dot stereograms for presenting 3D microscopic confocal images

Presenting 3D images produced by confocal optical slicing techniques in a static and easily publishable form can be difficult. Here, we demonstrate the presentation of the data as a single image random dot stereogram (SIRD), which can be viewed as a 3D object by 'defocusing' the eyes. The production of the SIRD employs three steps: (i) acquisition of the optical slices using confocal techniques, (ii) allocation of a suitable grey level to the object in each slice to provide depth-encoding information for the final image and (iii) calculation of the SIRD from the composite depth-encoded image. The technique is demonstrated with a limited number of optical slices through an acridine-orange-stained neutrophil (diameter =10 μm), in order to show the relative positions of the nuclear lobes in the cell.     

Three-dimensional imaging in double aberration-corrected scanning confocal electron microscopy, part I: elastic scattering

A transmission electron microscope fitted with both pre-specimen and post-specimen spherical aberration correctors enables the possibility of aberration-corrected scanning confocal electron microscopy. Imaging modes available in this configuration can make use of either elastically or inelastically scattered electrons. In this paper we consider image contrast for elastically scattered electrons. It is shown that there is no linear phase contrast in the confocal condition, leading to very low contrast for a single atom. Multislice simulations of a thicker crystalline sample show that sample vertical location and thickness can be accurately determined. However, buried impurity layers do not give strong, nor readily interpretable contrast. The accompanying paper examines the detection of inelastically scattered electrons in the confocal geometry.     

AFM/CLSM data visualization and comparison using an open-source toolkit

There is a vast difference in the traditional presentation of AFM data and confocal data. AFM data are presented as surface contours while confocal data are usually visualized using either surface- or volume-rendering techniques. Finding a common meaningful visualization platform is not an easy task. AFM and CLSM technologies are complementary and are more frequently being used to image common biological systems. In order to provide a presentation method that would assist us in evaluating cellular morphology, we propose a simple visualization strategy that is comparative, intuitive, and operates within an open-source environment of ImageJ, SurfaceJ, and VolumeJ applications. In order to find some common ground for AFM-CLSM image comparison, we have developed a plug-in for ImageJ, which allows us to import proprietary image data sets into this application. We propose to represent both AFM and CLSM image data sets as shaded elevation maps with color-coded height. This simple technique utilizes the open source VolumeJ and SurfaceJ plug-ins. To provide an example of this visualization technique, we evaluated the three-dimensional architecture of living chick dorsal root ganglia and sympathetic ganglia measured independently with AFM and CLSM.     

High-aperture cryogenic light microscopy

Living cells are highly organized in space and time, which makes spatially and temporally confined manipulations an indispensable tool in cell biology. Laser-based nanosurgery is an elegant method that allows precise ablation of intracellular structures. Here, we show cutting of fluorescently labelled microtubules and mitotic spindles in fission yeast, performed with a picosecond laser coupled to a confocal microscope. Diverse effects from photo-bleaching to partial and complete breakage are obtained by varying the exposure time, while simultaneously imaging the structures of interest. Using this system we developed an efficient technique to generate enucleated cells without perturbing the distribution of other organelles. This enucleation method can be used to study the cytoskeleton in a nucleus-free environment, as well as the role of the nucleus in cell growth and a variety of cellular functions.     

Data mining and statistical inference in selective laser melting

Selective laser melting (SLM) is an additive manufacturing process that builds a complex three-dimensional part, layer-by-layer, using a laser beam to fuse fine metal powder together. The design freedom afforded by SLM comes associated with complexity. As the physical phenomena occur over a broad range of length and time scales, the computational cost of modeling the process is high. At the same time, the large number of parameters that control the quality of a part make experiments expensive. In this paper, we describe ways in which we can use data mining and statistical inference techniques to intelligently combine simulations and experiments to build parts with desired properties. We start with a brief summary of prior work in finding process parameters for high-density parts. We then expand on this work to show how we can improve the approach by using feature selection techniques to identify important variables, data-driven surrogate models to reduce computational costs, improved sampling techniques to cover the design space adequately, and uncertainty analysis for statistical inference. Our results indicate that techniques from data mining and statistics can complement those from physical modeling to provide greater insight into complex processes such as selective laser melting.     

Theory of confocal fluorescence imaging in the programmable array microscope (PAM)

The programmable array microscope (PAM) uses a spatial light modulator (SLM) to generate an arbitrary pattern of conjugate illumination and detection elements. The SLM dissects the fluorescent light imaged by the objective into a focal conjugate image, I_c, formed by the ‘in-focus’ light, and a nonconjugate image, I_nc, formed by the ‘out-of-focus’ light. We discuss two different schemes for confocal imaging using the PAM. In the first, a grid of points is shifted to scan the complete image. The second, faster approach, uses a short tiled pseudorandom sequence of two-dimensional patterns. In the first case, I_c is analogous to a confocal image and I_nc to a conventional image minus I_c. In the second case I_c and I_nc are the sum and the difference, respectively, of a conventional and a confocal image. The pseudorandom sequence approach requires post-processing to retrieve the confocal part, but generates significantly higher signal levels for an equivalent integration time.     

Machined surface generation using wavelet filtering

The surface geometry of a workpiece after machining is a major concern in the industry. Thus, it is important to generate its geometry, reconfiguring and modifying it by a wavelet-based filtering technique with confocal laser scanning microscopy (CLSM). This technique is well-suited to construct the three-dimensional surface of a machined workpiece. The problem of noise on the surface always occurs when treating the raw height data (HEI: Height Encoded Image) acquired by CLSM. The noise occurs in constructing the surface height image (HEI) due to an electronic and photographic image process and must be filtered. In this paper, a wavelet-based filtering algorithm is used to eliminate noise in the shape features of the surface on a micro scale so as to realize an accurate geometry for further topographic analysis. The filtered surface patterns enable us to predict the machined surface and analyze surface characteristics in varied shapes of different levels. Also, to consider the tilting that inevitably occurs in mounting a specimen on the table, the surface is compensated and flattened in the x and y-directions simultaneously. This newly developed technique combines CLSM and wavelet filtering generates various micro machined surfaces over a wide range of surface resolutions on a micro scale     

Measurement of co-localization of objects in dual-colour confocal images

A method to measure the degree of co-localization of objects in confocal dual-colour images has been developed. This image analysis produced two coefficients that represent the fraction of co-localizing objects in each component of a dual-channel image. The generation of test objects with a Gaussian intensity distribution, at well-defined positions in both components of dual-channel images, allowed an accurate investigation of the reliability of the procedure. To do that, the co-localization coefficients were determined before degrading the image with background, cross-talk and Poisson noise. These synthesized sources of image deterioration represent sources of deterioration that must be dealt with in practical confocal imaging, namely dark current, non-specific binding and cross-reactivity of fluorescent probes, optical cross-talk and photon noise. The degraded images were restored by filtering and cross-talk correction. The co-localization coefficients of the restored images were not significantly different from those of the original undegraded images. Finally, we tested the procedure on images of real biological specimens. The results of these tests correspond with data found in the literature. We conclude that the co-localization coefficients can provide relevant quantitative information about the positional relation between biological objects or processes.     

Assembling and imaging of his-tag green fluorescent protein on mica surfaces studied by atomic force microscopy and fluorescence microscopy

The adsorption of his-tag green fluorescent protein (GFPH(6)) onto the mica surfaces has been studied by atomic force microscopy (AFM) and laser confocal fluorescence microscopy. By controlling the adsorption conditions, separated single GFPH(6) and GFPH(6) monolayer can be adsorbed and formed on mica surfaces. In present experiments, based on the AFM measurement, we found that the adsorbed GFPH(6) was bound on the mica surface with its beta-sheets. The formed GFPH(6) monolayer on mica surfaces was flat, uniform, and stable. Some applications of the formed monolayer have been demonstrated. The formed monolayer can be used as a substrate for DNA imaging and AFM mechanical lithography.     

Application of convolutional neural network to acquisition of clear images for objects with large vertical size in stereo light microscope vision system

For an object with large vertical size that exceeds the certain depth of a stereo light microscope (SLM), its image will be blurred. To obtain clear images, we proposed an image fusion method based on the convolutional neural network (CNN) for the microscopic image sequence. The CNN was designed to discriminate clear and blurred pixels in the source images according to the neighborhood information. To train the CNN, a training set that contained correctly labeled clear and blurred images was created from an open‐access database. The image sequence to be fused was aligned at first. The trained CNN was then used to measure the activity level of each pixel in the aligned source images. The fused image was obtained by taking the pixels with the highest activity levels in the source image sequence. The performance was evaluated using five microscopic image sequences. Compared with other two fusion methods, the proposed method obtained better performance in terms of both visual quality and objective assessment. It is suitable for fusion of the SLM image sequence.     

Algae through the looking glass

Microalgae are one of the most suitable subjects for testing the potentiality of light microscopy and image analysis, because of the size of single cells, their endogenous chromaticity, and their metabolic and physiological characteristics. Microscope observations and image analysis can use microalgal cells from lab cultures or collected from water bodies as model to investigate metabolic processes, behavior/reaction of cells under chemical or photic stimuli, and dynamics of population in the natural environment in response to changing conditions. In this paper we will describe the original microscope we set up together with the image processing techniques we improved to deal with these topics. Our system detects and recognizes in‐focus cells, extracts their features, measures cell concentration in multi‐algal samples, reconstructs swimming cell tracks, monitors metabolic processes, and measure absorption and fluorescent spectra of subcellular compartments. It can be used as digital microscopy station for algal cell biology and behavioral studies, and field analysis applications.     

Quantitative measurement of the resolution and sensitivity of confocal microscopes using line-scanning fluorescence correlation spectroscopy

Spatial resolution and the sensitivity to detect a fluorophore are the two most important optical parameters that characterize a confocal microscope. However, these are rather difficult to estimate quantitatively. We show that fluorescence correlation spectroscopy (FCS) provides an easy and reliable measure of these quantities. We modify existing schemes for performing FCS on a commercial confocal microscope to carry out these measurements, and provide an analysis routine that can yield the relevant quantities. Our method does not require any modification of the confocal microscope, yet it yields a robust measure of the resolution and sensitivity of the instrument.     

拉曼光谱成像技术新进展及其应用

拉曼光谱成像技术是拉曼光谱分析技术的新发展,借助于现代共焦显微拉曼光谱仪器以及新型信号探测装置,它把简单的单点分析方式拓展到对一定范围内样品进行综合分析,用图像的方式显示样品的化学成分空间分布、表面物理化学性质等更多信息。本文介绍拉曼光谱成像技术的基本原理和实验方法,并且特别介绍HORIBA Jobin Yvon公司的新型快速拉曼成像技术SWIFT和DuoScan,最后用实验实例说明这些技术的重要应用。     

A new method of imaging particle tracks in solid state nuclear track detectors

Solid state nuclear track detectors are used to determine the concentration of α particles in the environment. The standard method for assessing exposed detectors involves 2D image analysis. However 3D imaging has the potential to provide additional information relating to angle as well as to differentiate clustered hit sequences and possibly energy of α particles but this could be time consuming. Here we describe a new method for rapid high-resolution 3D imaging of solid state nuclear track detectors. A 'LEXT' OLS3100 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) was used in confocal mode to successfully obtain 3D image data on four CR-39 plastic detectors. Three-dimensional visualization and image analysis enabled characterization of track features. This method may provide a means of rapid and detailed 3D analysis of solid state nuclear track detectors.     

Biological imaging using fast laser scanning heterodyne differential phase confocal microscopes

Differential phase microscopy has proved invaluable in the study of live, unstained, thin biological samples because of its ability to image changes in refractive index and topography. Similarly, because of its optical sectioning capability, confocal microscopy is now a well-established technique in the study of relatively thick live biological samples. This paper describes the development and application of two differential phase heterodyne confocal microscopes, and compares their performance. The use of these systems for imaging in-vitro cell and tissue cultures is considered and compared with confocal reflection microscopy. It is demonstrated that the differential phase capability reveals subcellular structural information not readily seen in the confocal reflection images. This technique opens up the possibility of imaging thick unstained live tissues, avoiding cell damage and artefacts associated with staining procedures. Furthermore, the differential phase images can be used to provide a visual frame within which stained features can be located.     

Study on clear stereo image pair acquisition method for small objects with big vertical size in SLM vision system

Microscopic vision system with stereo light microscope (SLM) has been applied to surface profile measurement. If the vertical size of a small object exceeds the range of depth, its images will contain clear and fuzzy image regions. Hence, in order to obtain clear stereo images, we propose a microscopic sequence image fusion method which is suitable for SLM vision system. First, a solution to capture and align image sequence is designed, which outputs an aligning stereo images. Second, we decompose stereo image sequence by wavelet analysis theory, and obtain a series of high and low frequency coefficients with different resolutions. Then fused stereo images are output based on the high and low frequency coefficient fusion rules proposed in this article. The results show that Δw₁(Δw₂) and ΔZ of stereo images in a sequence have linear relationship. Hence, a procedure for image alignment is necessary before image fusion. In contrast with other image fusion methods, our method can output clear fused stereo images with better performance, which is suitable for SLM vision system, and very helpful for avoiding image fuzzy caused by big vertical size of small objects. Microsc. Res. Tech. 79:408–421, 2016. © 2016 Wiley Periodicals, Inc.     

用于提琴琴弦三维振动测量的高速摄影系统

分析了琴弦的亥姆霍兹运动,设计了一个基于高速摄影的非接触式光学测量系统,用于测量真实小提琴上琴弦的振动。通过特殊的光路设计和在琴弦上设置标定点,以7 000 frame/s的速度和336×480的图像分辨率拍摄了拉弦和拨弦时琴弦上标定点的三维振动形态。采用圆形霍夫变换图像处理算法、奇异值分解算法和自动批处理,对记录的大量序列图像进行处理,提取了弦振动的位移和轨迹等数据。实验结果表明,该测量系统能够精确跟踪小提琴琴弦的振动状态和包络轨迹,振动位移曲线的图像处理精确度达到0.03 mm。该系统为进一步研究提琴琴弦的振动机理和建立精确的琴弦振动理论模型提供了一种有效的实验方法。