Micro axial tomography: a miniaturized, versatile stage device to overcome resolution anisotropy in fluorescence light microscopy

With the development of novel fluorescence techniques, high resolution light microscopy has become a challenging technique for investigations of the three-dimensional (3D) micro-cosmos in cells and sub-cellular components. So far, all fluorescence microscopes applied for 3D imaging in biosciences show a spatially anisotropic point spread function resulting in an anisotropic optical resolution or point localization precision. To overcome this shortcoming, micro axial tomography was suggested which allows object tilting on the microscopic stage and leads to an improvement in localization precision and spatial resolution. Here, we present a miniaturized device which can be implemented in a motor driven microscope stage. The footprint of this device corresponds to a standard microscope slide. A special glass fiber can manually be adjusted in the object space of the microscope lens. A stepwise fiber rotation can be controlled by a miniaturized stepping motor incorporated into the device. By means of a special mounting device, test particles were fixed onto glass fibers, optically localized with high precision, and automatically rotated to obtain views from different perspective angles under which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopic equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives. Only very few images registered under different rotation angles are sufficient for full 3D reconstruction. The results indicate the principal advantage of the micro axial tomography approach for many microscopic setups therein and also those of improved resolutions as obtained by high precision localization determination.     

Transmission electron microtomography without the "missing wedge" for quantitative structural analysis

A three-dimensional (3D) visualization and structural analysis of a rod-shaped specimen of a zirconia/polymer nanocomposite material were carried out by transmission electron microtomography (TEMT) with particular emphasis on complete rotation of the specimen (tilt angular range: +/-90 degrees ). In order to achieve such an ideal experimental condition for the TEMT, improvements in the specimen as well as the sample holder were made. A rod-shaped specimen was necessary in order to obtain a high transmission of the specimen upon tilting to large angles. The image resolution of the reconstructed tomogram was isotropic, in sharp contrast to the anisotropic image resolution of the conventional TEMT with a limited angular range (the "missing wedge" problem). A volume fraction of zirconia, phi, evaluated from the 3D reconstruction was in quantitative agreement with the known composition of the nanocomposite. A series of 3D reconstructions was made from the tilt series with complete rotation by limiting the maximum tilt angle, alpha, from which a couple of structural parameters, the volume fraction and surface area per unit volume, Sigma, of the zirconia, were evaluated as a function of alpha. It was confirmed from actual experimental data that both phi and Sigma slightly decreased with the increasing alpha and reached constant values at around alpha=80 degrees , suggesting that the specimen may have to be tilted to +/-80 degrees for truly quantitative measurements.     

Angular reconstitution: a posteriori assignment of projection directions for 3D reconstruction

In computerized tomography as well as in most problems of three-dimensional reconstruction from projections, one knows from the experimental set-up the angular relationships between the projections from which the reconstruction is to be calculated. A serious difficulty is encountered when the angles are not known. In this paper, a method of "angular reconstitution" is described, which allows the a posteriori determination of the relative angular orientations of the projections and thus enables the three-dimensional reconstruction of the object to be calculated. For asymmetric objects, a minimum of three projections is required, which should not be related by a tilt around a single rotation axis. The method can be applied to determine the three-dimensional structure of biological macromolecules based on electron micrographs of randomly oriented individual molecules. Angular reconstitution, in combination with multivariate statistical techniques to classify and average the characteristic views of a molecule forms a complete, self-contained methodology for molecular structure analysis by electron microscopy.     

Precise 3D image alignment in micro-axial tomography

Micro (µ‐) axial tomography is a challenging technique in microscopy which improves quantitative imaging especially in cytogenetic applications by means of defined sample rotation under the microscope objective. The advantage of µ‐axial tomography is an effective improvement of the precision of distance measurements between point‐like objects. Under certain circumstances, the effective (3D) resolution can be improved by optimized acquisition depending on subsequent, multi‐perspective image recording of the same objects followed by reconstruction methods. This requires, however, a very precise alignment of the tilted views. We present a novel feature‐based image alignment method with a precision better than the full width at half maximum of the point spread function. The features are the positions (centres of gravity) of all fluorescent objects observed in the images (e.g. cell nuclei, fluorescent signals inside cell nuclei, fluorescent beads, etc.). Thus, real alignment precision depends on the localization precision of these objects. The method automatically determines the corresponding objects in subsequently tilted perspectives using a weighted bipartite graph. The optimum transformation function is computed in a least squares manner based on the coordinates of the centres of gravity of the matched objects. The theoretically feasible precision of the method was calculated using computer‐generated data and confirmed by tests on real image series obtained from data sets of 200 nm fluorescent nano‐particles. The advantages of the proposed algorithm are its speed and accuracy, which means that if enough objects are included, the real alignment precision is better than the axial localization precision of a single object. The alignment precision can be assessed directly from the algorithm's output. Thus, the method can be applied not only for image alignment and object matching in tilted view series in order to reconstruct (3D) images, but also to validate the experimental performance (e.g. mechanical precision of the tilting). In practice, the key application of the method is an improvement of the effective spatial (3D) resolution, because the well‐known spatial anisotropy in light microscopy can be overcome. This allows more precise distance measurements between point‐like objects.     

Reconstruction and display of curvilinear objects from optical section data using 3-D curve fitting algorithms

Biological objects resembling filaments are often highly elongated while presenting a small cross-sectional area. Examination of such objects requires acquisition of images from regions large enough to contain entire objects, but at sufficiently high resolution to resolve individual filaments. These requirements complicate the application of conventional optical sectioning and volume reconstruction techniques. For example, objective lenses used to acquire images of entire filaments or filament networks may lack sufficient depth ( Z ) resolution to localize filament cross-sections along the optical axis. Because volume reconstruction techniques consider only the information represented by a single volume element (voxel), views of filament networks reconstructed from images obtained at low Z -resolution will not accurately represent filament morphology. A possible solution to these problems is to simultaneously utilize all available information on the path of an object by fitting 3-D curves through data points localized in 2-D images. Here, we present an application of this approach to the reconstruction of microtubule networks from 2-D optical sections obtained using confocal microscopy, and to synthesized curves which have been distorted using a simple mathematical model of optical sectioning artefacts. Our results demonstrate that this strategy can produce high resolution 3-D views of filamentous objects from a small number of optical sections.     

Beyond the crystallization paradigm: structure determination from diffraction patterns from ensembles of randomly oriented particles

We amplify on the principles of the method we have recently proposed for recovering an oversampled diffraction pattern of a single particle from measured diffraction patterns from multiple particles in orientations related by rotation about an axis parallel to the incident radiation. We propose an alternative method of phasing a reference resolution ring by means of a non-negativity constraint on the diffraction intensities, point out the need for caution about enantiomeric ambiguities in the reconstruction of a diffraction pattern from its angular correlations, and show that converged correlations may be deduced by appropriate averaging of even very noisy data.     

3D reconstruction of histological sections: Application to mammary gland tissue

In this article, we present a novel method for the automatic 3D reconstruction of thick tissue blocks from 2D histological sections. The algorithm completes a high‐content (multiscale, multifeature) imaging system for simultaneous morphological and molecular analysis of thick tissue samples. This computer‐based system integrates image acquisition, annotation, registration, and three‐dimensional reconstruction. We present an experimental validation of this tool using both synthetic and real data. In particular, we present the 3D reconstruction of an entire mouse mammary gland and demonstrate the integration of high‐resolution molecular data. Microsc. Res. Tech. 73:1019–1029, 2010. © 2010 Wiley‐Liss, Inc.     

Three‐dimensional imaging of porous media using confocal laser scanning microscopy

In the last decade, imaging techniques capable of reconstructing three‐dimensional (3‐D) pore‐scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO₂ storage potential. CLSM has a unique capability of producing 3‐D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z‐dimension) that can be imaged in porous materials. In this study, we introduce a ‘grind and slice’ technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3‐D confocal volumetric data into pores and grains. Finally, we use the resulting 3‐D pore‐scale binarized confocal data obtained to quantitatively determine petrophysical pore‐scale properties such as total porosity, macro‐ and microporosity and single‐phase permeability using lattice Boltzmann (LB) simulations, validated by experiments.     

基于数字微镜器件的并行彩色共聚焦测量系统

彩色共聚焦技术因其高分辨率、高测速的特点,在表面形貌测量领域备受关注,然而现有的彩色共聚焦技术多为单点测量,一定程度上限制了测量效率。本文在彩色共聚焦技术的基础上,以DMD作为光分束器件,结合自主研发的大口径色散管镜,利用面阵彩色相机作为光电接收器件,研究和建立了基于数字微镜器件的并行彩色共聚焦实验平台,实现了对被测物面上多个探测点的并行图像处理。最终,利用所搭建的并行彩色共聚焦测量系统,对50μm高的台阶和自制台阶进行了测量,并对硬币的表面形貌进行了三维恢复。实验结果表明,该测量系统的轴向测量范围为300μm,测量精度达到微米级;同时,能够较好地恢复硬币的表面形貌特征,具有较好的测量效率与可靠性。     

Angular distribution of polarized photon-pairs in a scattering medium with a Zeeman laser scanning confocal microscope

A novel confocal microscope designed for use with turbid media is proposed. We use a Zeeman laser as the light source. Based on the properties of two‐frequency polarized photon‐pairs and the common‐path feature of polarized photon‐pairs with heterodyne detection employed in the proposed confocal microscope, three gatings (spatial filtering gating, polarization gating and spatial coherence gating) are thus simultaneously incorporated in the microscope. Experimental results for the angular distribution of polarized photon‐pairs in a scattering medium indicate that polarization gating and spatial coherence gating preclude the detection of multiply scattered photons, whereas the pinhole selects the least scattered photon‐pairs. Thus, better performance for axial resolution than can be obtained with a conventional confocal microscope is demonstrated experimentally. In addition, the proposed microscope is able to either look deeper into a turbid medium or work with a denser medium; furthermore, the axial resolution is improved.     

A novel approximation method of CTF amplitude correction for 3D single particle reconstruction

The typical resolution of three-dimensional reconstruction by cryo-EM single particle analysis is now being pushed up to and beyond the nanometer scale. Correction of the contrast transfer function (CTF) of electron microscopic images is essential for achieving such a high resolution. Various correction methods exist and are employed in popular reconstruction software packages. Here, we present a novel approximation method that corrects the amplitude modulation introduced by the contrast transfer function by convoluting the images with a piecewise continuous function. Our new approach can easily be implemented and incorporated into other packages. The implemented method yielded higher resolution reconstructions with data sets from both highly symmetric and asymmetric structures. It is an efficient alternative correction method that allows quick convergence of the 3D reconstruction and has a high tolerance for noisy images, thus easing a bottleneck in practical reconstruction of macromolecules.     

A software tool for tomographic axial superresolution in STED microscopy

A method for generating three‐dimensional tomograms from multiple three‐dimensional axial projections in STimulated Emission Depletion (STED) superresolution microscopy is introduced. Our STED< method, based on the use of a micromirror placed on top of a standard microscopic sample, is used to record a three‐dimensional projection at an oblique angle in relation to the main optical axis. Combining the STED< projection with the regular STED image into a single view by tomographic reconstruction, is shown to result in a tomogram with three‐to‐four‐fold improved apparent axial resolution. Registration of the different projections is based on the use of a mutual‐information histogram similarity metric. Fusion of the projections into a single view is based on Richardson‐Lucy iterative deconvolution algorithm, modified to work with multiple projections. Our tomographic reconstruction method is demonstrated to work with real biological STED superresolution images, including a data set with a limited signal‐to‐noise ratio (SNR); the reconstruction software (SuperTomo) and its source code will be released under BSD open‐source license.     

浅谈共聚焦显微技术

共聚焦显微镜以其高对比度、高分辨率及可重建三维图像的独特优势,在生物医学研究、微细加工、半导体和高分子材料的生产检测等领域获得广泛应用。常用的共聚焦技术方法有:传统的激光扫描共聚焦显微镜(LSCM),其特点是获得的图像对比度和分辨率高,但需要逐点扫描,帧成像时间长,系统复杂,体积大,价格昂贵;碟片共聚焦显微镜(SDCM)是采用多光束扫描的方法来获得共聚焦图像,速度可以大大提高,但牺牲了共聚焦图像的分辨率,系统更为复杂,且不能调整轴向分辨率;结构光显微镜(SIM)具有方法简单,可模块化设计,成本低,成像质量接近于激光扫描共聚焦显微镜,成像速度快,性价比较高。     

Three-dimensional optical microscopy using tilted views

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the x–y plane. This is true of conventional or confocal microscopes. To alleviate this problem we used multiple tilted views to supply the ‘missing data’ and thus increase the resolution in z. A special tilting stage was constructed which allowed specimens to be rotated through large angles. The relative, translation, rotation and z-spacing between data sets were determined by a novel Wiener/phase cross-correlation function. Once brought to a common coordinate system the data sets can be combined by Fourier space techniques similar to those used in X-ray crystallography. We applied this technique to metaphase chromosomes from intact embryos of Drosophila melanogaster. As determined from significant intensity in the Fourier transform, the resolution of the final reconstruction was about 0?25 μm in x and y, and 0?4 μm in z.     

Image reconstruction from sparse data in synchrotron-radiation-based microtomography

Synchrotron-radiation-based microcomputed-tomography (SR-μCT) is a powerful tool for yielding 3D structural information of high spatial and contrast resolution about a specimen preserved in its natural state. A large number of projection views are required currently for yielding SR-μCT images by use of existing algorithms without significant artifacts. When a wet biological specimen is imaged, synchrotron x-ray radiation from a large number of projection views can result in significant structural deformation within the specimen. A possible approach to reducing imaging time and specimen deformation is to decrease the number of projection views. In the work, using reconstruction algorithms developed recently for medical computed tomography (CT), we investigate and demonstrate image reconstruction from sparse-view data acquired in SR-μCT. Numerical results of our study suggest that images of practical value can be obtained from data acquired at a number of projection views significantly lower than those used currently in a typical SR-μCT imaging experiment.     

Isotropic high-resolution three-dimensional confocal micro-rotation imaging for non-adherent living cells

Recently, micro-rotation confocal microscopy has enabled the acquisition of a sequence of micro-rotated images of nonadherent living cells obtained during a partially controlled rotation movement of the cell through the focal plane. Although we are now able to estimate the three-dimensional position of every optical section with respect to the cell frame, the reconstruction of the cell from the positioned micro-rotated images remains a last task that this paper addresses. This is not strictly an interpolation problem since a micro-rotated image is a convoluted two-dimensional map of a three-dimensional reality. It is rather a 'reconstruction from projection' problem where the term projection is associated to the PSF of the deconvolution process. Micro-rotation microscopy has a specific difficulty. It does not yield a complete coverage of the volume. In this paper, experiments illustrate the ability of the classical EM algorithm to deconvolve efficiently cell volume despite of the incomplete coverage. This cell reconstruction method is compared to a kernel-based method of interpolation, which does not take account explicitly the point-spread-function (PSF). It is also compared to the standard volume obtained from a conventional z-stack. Our results suggest that deconvolution of micro-rotation image series opens some exciting new avenues for further analysis, ultimately laying the way towards establishing an enhanced resolution 3D light microscopy.     

基于多图像融合的MEMS显微三维形貌重构

针对微型机电系统(MEMS)的三维测量,显微镜或光学轮廓干涉仪等传统方法存在显微测量精度低、设备成本高等问题,且当结构含有较多断裂面时,解包裹算法效果欠佳。本文提出一种基于多图像融合的MEMS显微三维测量方法。不同于多角度显微三维测量方法,本研究首先利用单目显微镜,通过单一轴向移动获取一系列测量目标深度信息的单一角度图像,并利用去雾算法对图像进行预处理,实现了去噪和有效信息提取的目的;然后通过聚焦测度算法获取待测对象的深度信息;最后利用数据处理软件进行三维拟合。基于上述原理,本文以焦平面阵列(FPA)作为待测目标进行了测量实验。本文提出的三维测量方法和图像处理算法可获得更准确的FPA形貌,可清晰显示反射面与支腿部分及反射面上的释放孔,测得FPA的支腿长度为110.6μm,每个反射面的像元尺寸为120.8μm×70.8μm,与设计值基本吻合,解决了断裂面难以测量的问题,同时降低了微结构测量的难度和成本。单目显微镜单向移动的多图像融合测量技术对MEMS的三维形貌测量具有重要意义,去雾算法在图像融合与三维测量的图像处理也有很好的应用价值。     

一种多视图的视图分离和视图关系确立方法

提出一种多视图分离和视图关系确立方法,在分析线条连通集合以及线条连通集合之间包容关系的基础上,进行视图分离;根据制图规划以及图形标注信息建立视图之间的毗邻关系;较好地解决了包括位置任意布的斜视图、局部视图在内的机械零件图多视图的视图分割和视图关系确立问题,为后续图形信息高层理解提供了良好的基础。     

MRT letter: A unified accelerated maximum likelihood technique for widefield,confocal, and super‐resolution 4Pi microscopy

We propose an algorithmic technique for accelerating maximum likelihood (ML) algorithm for image reconstruction in fluorescence microscopy. This is made possible by integrating Biggs–Andrews (BA) method with ML approach. The results on widefield, confocal, and super‐resolution 4Pi microscopy reveal substantial improvement in the speed of 3D image reconstruction (the number of iterations has reduced by approximately one‐half). Moreover, the quality of reconstruction obtained using accelerated ML closely resembles with nonaccelerated ML method. The proposed technique is a step closer to realize real‐time reconstruction in 3D fluorescence microscopy. Microsc. Res. Tech. 78:331–335, 2015. © 2015 Wiley Periodicals, Inc.     

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.