A modified phasor approach for analyzing time-gated fluorescence lifetime images

Fluorescence lifetime imaging is a versatile tool that permits mapping the biochemical environment in the cell. Among various fluorescence lifetime imaging techniques, time-correlated single photon counting and time-gating methods have been demonstrated to be very efficient and robust for the imaging of biological specimens. Recently, the phasor representation of lifetime images became popular because it provides an intuitive graphical view of the fluorescence lifetime content of the images and, when used for global analysis, significantly improves the overall S/N of lifetime analysis. Compared to time-correlated single photon counting, time gating methods can provide higher count rates (～10 MHz) but at the cost of truncating and under sampling the decay curve due to the limited number of gates commonly used. These limitations also complicate the implementation of the phasor analysis for time-gated data. In this work, we propose and validate a theoretical framework that overcomes these problems. This modified approach is tested on both simulated lifetime images and on cells. We demonstrate that this method is able to retrieve two lifetimes from time gating data that cannot be resolved using standard (non-global) fitting techniques. The new approach increases the information that can be obtained from typical measurements and simplifies the analysis of fluorescence lifetime imaging data.     

Multimodal and non‐linear optical microscopy applications in reproductive biology

A plethora of optical techniques is currently available to obtain non‐destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four‐dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567–582, 2016. © 2016 Wiley Periodicals, Inc.     

Retrieval of object information by inverse problems in electron diffraction

The imaging of crystal defects by high-resolution transmission electron microscopy or with the help of the electron diffraction contrast technique is well known and routinely used. However, a direct and phenomenological analysis of electron micrographs is mostly not possible, but requires the application of image simulation and matching techniques. The trial-and-error matching technique is the indirect solution to the direct scattering problem applied to analyse the nature of the object under investigation. Alternatively, inverse problems as direct solutions of electron scattering equations can be deduced using either an invertible linearized eigenvalue system or a discretized form of the diffraction equations. This analysis is based on the knowledge of the complex electron wave at the exit plane of an object reconstructed for the surrounding of single reflections by electron holography or other wave reconstruction techniques. In principle, it enables directly the retrieval of the local thickness and orientation of a sample as well as the refinement of potential coefficients or the determination of the atomic displacements, caused by a crystal lattice defect, relative to the atom positions of the perfect lattice. Considering especially the sample orientation as perturbation the solution is given by a generalized and regularized Moore–Penrose inverse, where the resulting numerical algorithms imply ill-posed inverse problems.     

数据预处理技术和机器学习方法在质子转移反应质谱中的应用

质子转移反应质谱（PTR-MS）法是一种用于检测挥发性有机物（VOCs）的分析技术。它具有检测限低、响应速度快、无需样品前处理、实时分析等特点,在大气化学、环境化学、食品、生物医学等领域得到广泛应用。随着PTR-MS应用的扩展和样品种类的增加,如何从复杂的质谱数据中提取特征,并寻找内在规律,对分析算法的处理能力提出了更高的要求。本工作从数据预处理技术和机器学习方法两方面展开论述,归纳了具有PTR-MS特点的数据预处理技术,总结了不同机器学习算法在PTR-MS数据分析中的应用,并讨论了它们的优点和不足。     

Increasing the reliability of the detection of flaws in complex structures by integrating the information of multiparametric nondestructive testing

The basic principles and methods for integrating information from multiparametric nondestructive testing for the purpose of detecting anomalies, flaws, and damage in different objects and structures are considered. Some experimental data and the results of the development of algorithms for the integration of nondestructive testing techniques that are aimed at solving formulated problems on the basis of telescoping-effect estimation methods and the construction of the multidimensional analogues of inverse probability methods are cited.     

计算成像技术在光学检测领域的研究进展

成像系统在利用传感器记录图像信息时,只记录了强度信息而丢失了重要的相位信息。 传统的干涉相位恢复技术由于 需要满足严格的干涉条件,在使用过程中受到一定限制。 随着计算成像技术的蓬勃发展,以强度传输方程和相干衍射成像以及 在此基础上发展起来的相干调制成像、叠层扫描相干衍射成像为代表的非干涉相位恢复技术受到广泛关注,并被运用于光学检 测领域。 这类技术无需参考光,系统结构简单,可通过衍射强度图直接获得相位信息,在检测领域有巨大的应用潜力。 基于此, 介绍了几种典型的计算成像技术的研究现状与最新进展,同时讨论分析了各类方法的主要技术特点。     

A Thomson parabola ion imaging spectrometer designed to probe relativistic intensity ionization dynamics of nanoclusters

Conventional techniques of probing ionization dynamics at relativistic intensities for extended target systems such as clusters are difficult both due to problems of achieving good charge resolution and signal integration over the focal volume. Simultaneous measurement of arrival time, necessary for these systems, has normally involved complicated methods. We designed and developed a Thomson parabola imaging spectrometer that overcomes these problems. Intensity sampling method evolved in this report is proved to be mandatory for probing ionization dynamics of clusters at relativistic intensities. We use this method to measure charge resolved kinetic energy spectra of argon nanoclusters at intensities of 4 × 10(18) W cm(-2).     

Flow monitoring by microwave imaging inside a cuboid cavity: Theory and numerical feasibility analysis

In this paper, we investigate multiphase flow monitoring inside metallic pipes by qualitative microwave imaging, where foreign objects inside the liquid flow are monitored to eliminate contamination. The motivation stems from the fact that conventional tomographic approaches of microwave imaging are inherently not suitable for real-time flow monitoring due to their computational requirements as these techniques aim to retrieve the dielectric permittivity profile inside the pipeline. In this context, we envision that the factorization method of qualitative inverse scattering theory, which is a shape retrieval algorithm from scattered field measurements, provides a better alternative for flow monitoring. To demonstrate the feasibility of such an approach, we first reformulate the factorization method by considering dyadic Green’s functions inside a cuboid cavity with perfectly conducting walls. Later, the formulation is simplified for a specific microwave measurement configuration to accelerate the reconstructions for real-time flow monitoring. Finally, flow monitoring is realized as a differential imaging procedure where foreign objects are detected by using differences of multi-static scattering parameters that are measured consecutively. The simulation studies performed for food and petroleum flows reveal the capabilities of the technique as a viable solution for different industrial flows.     

Fluorescence bleach rate imaging

Bleach rate imaging on a (cooled) CCD can be easily achieved using a confocal microscope with bilateral scanning and detection coupled to a workstation; it is as easy as acquiring regular fluorescence images. Several analysis and display methods for bleach rate imaging are presented such as the bleach map (and its inverse) and a matrix-based decomposition method for multi-labelled specimens based on the bleach rate differences between the dyes used. With these tools, bleach-rate-based imaging can become a viable alternative to multiple labelling techniques for component identification in fluorescent specimens.     

Quantification of micrometre-sized porosity in quasicrystals using coherent synchrotron radiation imaging

The ability to image phase distributions with high spatial resolution is a key capability of microscopy systems. Consequently, the development and use of phase microscopy has been an important aspect of microscopy research and development. Most phase microscopy is based on a form of interference. Some phase imaging techniques, such as differential interference microscopy or phase microscopy, have a low coherence requirement, which enables high‐resolution imaging but in effect prevents the acquisition of quantitative phase information. These techniques are therefore used mainly for phase visualization. On the other hand, interference microscopy and holography are able to yield quantitative phase measurements but cannot offer the highest resolution. A new approach to phase microscopy, quantitative phase‐amplitude microscopy (QPAM) has recently been proposed that relies on observing the manner in which intensity images change with small defocuses and using these intensity changes to recover the phase. The method is easily understood when an object is thin, meaning its thickness is much less than the depth of field of the imaging system. However, in practice, objects will not often be thin, leading to the question of what precisely is being measured when QPAM is applied to a thick object. The optical transfer function formalism previously developed uses three‐dimensional (3D) optical transfer functions under the Born approximation. In this paper we use the 3D optical transfer function approach of Streibl not for the analysis of 3D imaging methods, such as tomography, but rather for the problem of analysing 2D phase images of thick objects. We go on to test the theoretical predictions experimentally. The two are found to be in excellent agreement and we show that the 3D imaging properties of QPAM can be reliably predicted using the optical transfer function formalism.     

Image formation in scanned heterodyne microscope systems

Several new scanning microscopic techniques have recently been developed which rely on modulation of the optical beam to enhance the imaging performance. These systems have the common feature that the image is formed by detection or demodulation of an a.c. signal. Techniques have been developed which are sensitive to both intensity and phase information in the sample. We refer to such microscopes as heterodyne imaging systems (and by analogy single-frequency microscopes, operating at d.c. only, are referred to as homodyne) although as we will point out this term is only strictly applicable to the interferometric-based methods. Although the theory for the conventional homodyne scanning optical microscope is well developed there has been no systematic study of heterodyne microscopic techniques, which is the purpose of this paper. Several techniques are discussed and compared and the different ways of extracting amplitude and phase information are considered in detail with reference to experimental systems which have been demonstrated to have good imaging performance.     

The microcomputer and image analysis in diagnostic pathology.

This paper presents a snapshot view of the influence and direction of microcomputer technology for image analysis techniques in diagnostic pathology. Microcomputers have had considerable impact in bringing image analysis to wider application. Semi-automated tracing techniques are a simple means of providing objective data and assist in a wide range of diagnostic problems. From the common theme of reducing subjectivity in diagnostic assessment, an extensive body of research has accrued. Some studies have addressed the need for quality control for reliable, routine application. Video digitizer cards bring digital image analysis within the reach of laboratory budgets, providing powerful tools for investigation of a wide range of cellular and tissue features. The use of staining procedures compatible with quantitative evaluation has become equally important. As well as assisting scene segmentation, cytochemical and immunochemical staining techniques relate the data to biological processes. With the present state of the art, practical use of microcomputer based image analysis is impaired by limitations of information extraction and specimen throughput. Recent advances in colour video imaging provide an extra dimension in the analysis of multi-spectral stains. Improvements will also be felt with predictable increase in speed of microprocessors, and with single chip devices which deliver video rate processing. If the full potential of this hardware is realized, high-speed, routine analysis becomes feasible. In addition, a microcomputer imaging system can play host to companion functions, such as image archiving and transmission. With this outlook, the use of microcomputers for image analysis in diagnostic pathology is certain to increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenomenological and algorithmic methods for the solution of inverse problems of electromagnetic testing

Modern methods for the solution of inverse problems of nondestructive testing are described. The discussion is mainly focused on the methods based on a mathematical model of the respective physical phenomenon (so-called phenomenological methods) and the methods based on the algorithms for the analysis of digital signals (so-called algorithmic methods). The phenomenological methods involving a mathematical model assume that the configuration of the flaws in a tested specimen is varied until the norm of the mismatch between the model solution and the experimentally obtained signal is minimized. A good result is only guaranteed if the physics of the phenomenon in the model is close to reality. In algorithmic methods, the inversion procedure applied to experimental data is considered as an image-recognition problem. In this case, the signal is identified as a representative of the classes associated with known types of flaws. The classification algorithms, which are most frequently used for electromagnetic testing, are developed through identification of diagnostic signatures. This approach assumes the use of an artificial neural network trained with the signals from a predefined database that corresponds to a broad variety of flaws.     

Autofluorescence imaging, an excellent tool for comparative morphology

Here we present a set of methods for documenting (exo-)morphology by applying autofluorescence imaging. For arthropods, but also for other taxa, autofluorescence imaging combined with composite imaging is a fast documentation method with high-resolution capacities. Compared to conventional micro- and macrophotography, the illumination is much more homogenous, and structures are often better contrasted. Applying different wavelengths to the same object can additionally be used to enhance distinct structures. Autofluorescence imaging can be applied to dried and embedded specimens, but also directly on specimens within their storage liquid. This has an enormous potential for the documentation of rare specimens and especially type specimens without the need of preparation. Also for various fossils, autofluorescence can be used to enhance the contrast between the fossil and the matrix significantly, making even smallest details visible. 'Life-colour' fluorescence especially is identified as a technique with great potential. It provides additional information for which otherwise more complex methods would have to be applied. The complete range of differences and variations between fluorescence macrophotography and different types of fluorescence microscopy techniques are here explored and evaluated in detail. Also future improvements are suggested. In summary, autofluorescence imaging is a powerful, easy and fast-to-apply tool for morphological studies.     

Charge defects glowing in the dark

We investigate the effects of local charge defects in HREM imaging, using electron densities calculated by density functional methods. As a model of a planar interface with a local charge defect we use the polar MgO square root (111)-3 x 3R30 degrees surface, which has an additional hole per surface unit cell. A complimentary example, the non-polar MgO (100) surface that has no local charge defect is simulated for comparison. We show that the contrast due to local charge defects is rather high, and suggest that they should be directly observable.     

Three‐dimensional imaging of whole mouse models: comparing nondestructive X‐ray phase‐contrast micro‐CT with cryotome‐based planar epi‐illumination imaging

In this study, we compare two evolving techniques for obtaining high‐resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome‐based planar epi‐illumination imaging (cryo‐imaging). On the other hand, we examine X‐ray phase‐contrast micro‐computed tomography (micro‐CT) using synchrotron radiation. Cryo‐imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo‐frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X‐ray phase‐contrast imaging is based on the minute refraction of X‐rays inside the specimen and features higher soft‐tissue contrast than conventional, attenuation‐based micro‐CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft‐tissue contrast is strongly enhanced in the X‐ray phase images compared to the attenuation‐based images. This identifies phase‐contrast micro‐CT as a powerful tool for the study of small animal disease models.     

Image restoration for TV‐scan moving images acquired through a semiconductor backscattered electron detector

A semiconductor backscattered electron (BSE) detector has become popular in scanning electron microscopy session. However, detectors of semiconductor type have a serious disadvantage on the frequency characteristics. As a result, fast scan (e.g. TV‐scan) BSE image should be blurred remarkably. It is the purpose of this study to restore this degradation by using digital image processing technology. In order to improve it practically, we have to settle several problems, such as noise, undesirable processing artifacts, and ease of use. Image processing techniques in an impromptu manner like a conventional mask processing are unhelpful for this study, because a complicated degradation of output signal affects severely the phase response as well as the amplitude response of our SEM system. Hence, based on the characteristics of an SEM signal obtained from the semiconductor BSE detector, a proper inverse filter in Fourier domain is designed successfully. Finally, the inverse filter is converted to a special convolution mask, which is skillfully designed, and applied for TV‐scan moving BSE images. The improved BSE image is very effective in the work for finding important objects. SCANNING 31: 229–235, 2009. © 2010 Wiley Periodicals, Inc.