Image processing: a non-destructive method for measuring growth in cell and tissue culture

The use of machine vision in evaluating callus growth directly in Petri dishes is reported for carrot cell suspension growing in the presence of increasing concentrations of various herbicides. Machine vision was much more accurate and sensitive than visual counting of the cell aggregates. Image processing also gives more detailed information than does counting because of its ability to distinguish between intermediate levels of callus growth.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - MS Murashige and Skoog - GR Gelrite     

Differentiation of granulocytes in Pappenheim stained blood cell smears using standardized cytophotometric analysis

The well-known problems of the low reproducibility of peripheral blood smear analysis have for some time stimulated endeavours to automate blood cell classification. In the cytophotometric standardized color measurement and analysis, the computed color characteristics for the first time refer to an internationally accepted color system, allowing not only an international comparison of the computer color measurements but an unproblematic mutual interchange of color information between man and machine based on both the human visual color impressions and the conventional morphological color attributes of the white blood cells. The discriminatory power of the method is demonstrated by differentiating the cytoplasm granulations in basophil, eosinophil and neutrophil granulocytes.     

Parallel processing and image analysis in the eyes of mantis shrimps

The compound eyes of mantis shrimps, a group of tropical marine crustaceans, incorporate principles of serial and parallel processing of visual information that may be applicable to artificial imaging systems. Their eyes include numerous specializations for analysis of the spectral and polarizational properties of light, and include more photoreceptor classes for analysis of ultraviolet light, color, and polarization than occur in any other known visual system. This is possible because receptors in different regions of the eye are anatomically diverse and incorporate unusual structural features, such as spectral filters, not seen in other compound eyes. Unlike eyes of most other animals, eyes of mantis shrimps must move to acquire some types of visual information and to integrate color and polarization with spatial vision. Information leaving the retina appears to be processed into numerous parallel data streams leading into the central nervous system, greatly reducing the analytical requirements at higher levels. Many of these unusual features of mantis shrimp vision may inspire new sensor designs for machine vision.     

Analysis of morphological changes as a key method in studying psychiatric animal models

A major interest in the analysis of animal models of psychiatric diseases is their underlying cellular pathology and to gain information regarding whether pharmacological treatments, genetic differences or an altered environment exert an impact upon the brain morphology or on the morphology or activity of single neurones. In this review, several key methods will be introduced that allow the analysis of morphological changes that are frequently observed in psychiatric animal models. An overview of the techniques that enable dendritic arborisation, alterations in dendritic spines and changes in fibre densities to be analysed are described. Moreover, methods for the analysis of adult neurogenesis and neurodegeneration and for the analysis of neuronal activity in fixed brain tissue are described. An important step during the analysis of morphological changes is the estimation of the number of stained cells. Since conventional cell counting methods have several limitations, two different approaches that permit an estimate of the number of stained cells within three-dimensional tissue are also discussed.     

Vision without Frames: A Semiotic Paradigm of Event Based Computer Vision

Conventional imagers and almost all vision processes use and rely on theories that are based on the principle of static image-frames. A frame is a 2D matrix that represents the spatial locations of intensities of a scene projected on the imager. The notion of a frame itself is so embedded in machine vision, that it is usually taken for granted that this is how biological systems store light information. This paper presents a biosinpired event-based image formation principle, which output data rely on an asynchronous acquisition process. The generated information is stored in temporal volumes, which size and information depend only on the dynamic content of observed scenes. Practical analysis of such information will shows that the processing of visual information can only be based on a semiotic process. The paper also provides a general definition of the notion of visual features as the interpretation of signs according to different possible readings of the codified visual signal.     

Labour-Efficient In Vitro Lymphocyte Population Tracking and Fate Prediction Using Automation and Manual Review

Interest in cell heterogeneity and differentiation has recently led to increased use of time-lapse microscopy. Previous studies have shown that cell fate may be determined well in advance of the event. We used a mixture of automation and manual review of time-lapse live cell imaging to track the positions, contours, divisions, deaths and lineage of 44 B-lymphocyte founders and their 631 progeny in vitro over a period of 108 hours. Using this data to train a Support Vector Machine classifier, we were retrospectively able to predict the fates of individual lymphocytes with more than 90% accuracy, using only time-lapse imaging captured prior to mitosis or death of 90% of all cells. The motivation for this paper is to explore the impact of labour-efficient assistive software tools that allow larger and more ambitious live-cell time-lapse microscopy studies. After training on this data, we show that machine learning methods can be used for realtime prediction of individual cell fates. These techniques could lead to realtime cell culture segregation for purposes such as phenotype screening. We were able to produce a large volume of data with less effort than previously reported, due to the image processing, computer vision, tracking and human-computer interaction tools used. We describe the workflow of the software-assisted experiments and the graphical interfaces that were needed. To validate our results we used our methods to reproduce a variety of published data about lymphocyte populations and behaviour. We also make all our data publicly available, including a large quantity of lymphocyte spatio-temporal dynamics and related lineage information.     

Pupillometric analysis for assessment of gene therapy in Leber Congenital Amaurosis patients

ABSTRACT: BACKGROUND: Objective techniques to assess the amelioration of vision in patients with impaired visual function are needed to standardize efficacy assessment in gene therapy trials for ocular diseases. Pupillometry has been investigated in several diseases in order to provide objective information about the visual reflex pathway and has been adopted to quantify visual impairment in patients with Leber Congenital Amaurosis (LCA). In this paper, we describe detailed methods of pupillometric analysis and a case study on three Italian patients affected by Leber Congenital Amaurosis (LCA) involved in a gene therapy clinical trial at two follow-up time-points: 1 year and 3 years after therapy administration. METHODS: Pupillary light reflexes (PLR) were measured in patients who had received a unilateral subretinal injection in a clinical gene therapy trial. Pupil images were recorded simultaneously in both eyes with a commercial pupillometer and related software. A program was generated with MATLAB software in order to enable enhanced pupil detection with revision of the acquired images (correcting aberrations due to the inability of these severely visually impaired patients to fixate), and computation of the pupillometric parameters for each stimulus. Pupil detection was performed through Hough Transform and a non-parametric paired statistical test was adopted for comparison. RESULTS: The developed program provided correct pupil detection also for frames in which the pupil is not totally visible. Moreover, it provided an automatic computation of the pupillometric parameters for each stimulus and enabled semi-automatic revision of computerized detection, eliminating the need for the user to manually check frame by frame. With reference to the case study, the amplitude of pupillary constriction and the constriction velocity were increased in the right (treated eye) compared to the left (untreated) eye at both follow-up time-points, showing stability of the improved PLR in the treated eye. CONCLUSIONS: Our method streamlined the pupillometric analyses and allowed rapid statistical analysis of a range of parameters associated with PLR. The results confirm that pupillometry is a useful objective measure for the assessment of therapeutic effect of gene therapy in patients with LCA. Trial registration ClinicalTrials.gov NCT00516477.     

Haematopoietic cell clusters quantification using image analysis

Automatising the analysis of haematopoietic cells culture is a step which is necessary in order to develop the use of this kind of measurement in toxicological assessment. The purpose is the classification of cell aggregates into three groups namely micro-clusters, macro-clusters and colonies. This is classically done using human vision. However, reproducibility is not good and comparisons between experts or laboratories remains very difficult. In this work, we propose a method based on learning machine: different parameters representative of these three kinds of clusters are tested in order to choose the best discriminative ones, then a learning database is created in order to parametrise the software, before using it on actual clusters images. The algorithm used is the support vector machine (SVM). Results show an excellent ability because almost 90% of the test database is well classified using three geometric parameters namely area, minimum and maximum centroid distances.     

基于机器学习的风景园林智能化分析应用研究

机器学习使实现数据的智能化处理及充分利用数据中蕴含的知识与价值成为可能。探索基于机器学习在风景园林领域智能化分析应用的途径,开展3个实验。其中2个与数据分析研究相关,提出基于调研图像色彩聚类分析的城市色彩印象和基于图像识别技术的景观视觉质量评估与网络应用平台部署实验。最后1个实验与数字化设计创作相关,提出用于设计方案遴选的地形生成方法,包括2个子项目：应用深度学习生成对抗网络（GAN）的地形生成和建立遮罩、预测未知区域的高程。3个实验应用到机器学习中分类、聚类和回归3个主要方向中的算法以及深度学习的生成对抗网络,对传统的研究问题提出了基于机器学习新的研究方法。因此,在应用机器学习风景园林领域,可以有效地从多源数据中学习相互增强的知识,发现问题,并提出解决问题的新方法。     

Visual feedback is not necessary for the learning of novel dynamics

Background

When learning to perform a novel sensorimotor task, humans integrate multi-modal sensory feedback such as vision and proprioception in order to make the appropriate adjustments to successfully complete the task. Sensory feedback is used both during movement to control and correct the current movement, and to update the feed-forward motor command for subsequent movements. Previous work has shown that adaptation to stable dynamics is possible without visual feedback. However, it is not clear to what degree visual information during movement contributes to this learning or whether it is essential to the development of an internal model or impedance controller.

Methodology/Principle Findings

We examined the effects of the removal of visual feedback during movement on the learning of both stable and unstable dynamics in comparison with the case when both vision and proprioception are available. Subjects were able to learn to make smooth movements in both types of novel dynamics after learning with or without visual feedback. By examining the endpoint stiffness and force after learning it could be shown that subjects adapted to both types of dynamics in the same way whether they were provided with visual feedback of their trajectory or not. The main effects of visual feedback were to increase the success rate of movements, slightly straighten the path, and significantly reduce variability near the end of the movement.

Conclusions/Significance

These findings suggest that visual feedback of the hand during movement is not necessary for the adaptation to either stable or unstable novel dynamics. Instead vision appears to be used to fine-tune corrections of hand trajectory at the end of reaching movements.     

Morphological control and mensuration of potato plantlets from tissue cultures for automated micropropagation

Automation in plant micropropagation can be greatly simplified if the propagated plantlets have some morphological properties that facilitate automatic chopping and subsequent inspection and classification of the pre-cut plantlet segments by machine vision as viable propagules. We were able to control the morphogenic pattern of in vitro-propagated potato plantlets by adding various concentrations of ancymidol to the nutrient solution. It was found that plantlets cultured in 0.25 mg l^–1 ancymidol best fit the requirements for automated mass micropropagation; the mean internode length was sufficiently large (9–10 mm), the color contrast between leaves and stems was significantly enhanced, the stem was thicker than in the control treatment and the number of axillary buds per plantlet was maximized. Microtuber formation on segments isolated from plants cultured in 0.25 and 0.5 mg l^–1 ancymidol media was enhanced shortly after transfer to tuber induction medium in vitro. On shoot segments from control plants, microtuber formation started after 24–28 days.Machine vision was used to evaluate the morphological and color changes in cultured potato plants. Geometrical and color features such as the number of buds, internode length and color contrast between leaf and stem were precisely measured and automatically logged. Features were measured that till now could only be observed qualitatively.Abbreviations F/W fresh weight - RGB red, green, blue principal color components - VTR video tape recorder     

Direct quantification of in vitro cell growth through image analysis

Summary Objective, accurate, non-intrusive measurement of in vitro cell growth was realized through microcomputerized video image analysis. Recently-released video and digitizing hardware and software were incorporated into an analytical system which accurately quantified visual differences between cultures on a cell number or fresh mass basis. Sequential measurements during culture incubation further detected and quantified subtle changes in colony area and density resulting from growth. Each measurement was acquired rapidly, without encroaching on the in vitro environment, so cell growth was undisturbed. Custom software routines coordinated the quantification of this detailed record into precise cumulative growth curves.     

Computational methods for ultrastructural analysis of synaptic complexes

Electron microscopy (EM) provided fundamental insights about the ultrastructure of neuronal synapses. The large amount of information present in the contemporary EM datasets precludes a thorough assessment by visual inspection alone, thus requiring computational methods for the analysis of the data. Here, I review image processing software methods ranging from membrane tracing in large volume datasets to high resolution structures of synaptic complexes. Particular attention is payed to molecular level analysis provided by recent cryo-electron microscopy and tomography methods.     

A multivariate analysis of temporal change in Arikara craniometrics: A methodological approach

We have examined temporal changes in the cranial architecture of Arikara Amerindians from five archaeological sites in South Dakota which span a time period of approximately 230 years (ca. A.D. 1600–1830). We have utilized a multivariate statistical method based on a principal components analysis of the pooled within-groups correlation matrix rather than the more traditional methods of ascertaining morphological relationships, e.g., discriminant functions, Mahalanobis' D², or Penrose's Size and Shape. Our component structure, based on a regional sample and the mathematically simpler principal components analysis, is very similar to the factor structure obtained by Howells (1973) using a world-wide sample and factor analysis proper. This supports the notion of the “universality” of cranial structure. An axis of temporal variation was introduced into the component space by means of multiple regression. This analysis indicates that a substantial portion of the intergroup variation is temporal in nature and that systematic temporal changes occur along the facial height, transverse frontal flatness, and frontal profile flatness components. Earlier analyses of the same material by more conventional methods either did not detect the temporal trends at all or failed to isolate the specific nature of the temporal changes. The success of the present analysis attests to the value of examining morphological relationships by means of principal components.     

Diversity of Ca2+-induced morphology revealed by morphological phenotyping of Ca2+-sensitive mutants of Saccharomyces cerevisiae

Yeast cell morphology can be treated as a quantitative trait using the image processing software CalMorph. In the present study, we investigated Ca(2+)-induced morphological changes in Ca(2+)-sensitive (cls) mutants of Saccharomyces cerevisiae, based on the discovery that the characteristic Ca(2+)-induced morphological changes in the Ca(2+)-sensitive mutant zds1 reflect changes in the Ca(2+) signaling-mediated cell cycle control pathway. By applying hierarchical cluster analysis to the quantitative morphological data of 58 cls mutants, 31 of these mutants were classified into seven classes based on morphological similarities. The patterns of morphological change induced by Ca(2+) in one class differed from those of another class. Based on the results obtained using versatile methods for phenotypic analysis, we conclude that a high concentration of Ca(2+) exerts a wide variety of effects on yeast and that there are multiple Ca(2+)-regulatory pathways that are distinct from the Zds1p-related pathway.     

A mutation in the silver gene leads to defects in melanosome biogenesis and alterations in the visual system in the zebrafish mutant fading vision

Forward genetic screens have been instrumental in defining molecular components of visual function. The zebrafish mutant fading vision (fdv) has been identified in such a screen due to defects in vision accompanied by hypopigmentation in the retinal pigment epithelium (RPE) and body melanocytes. The RPE forms the outer most layer of the retina, and its function is essential for vision. In fdv mutant larvae, the outer segments of photoreceptors are strongly reduced in length or absent due to defects in RPE cells. Ultrastructural analysis of RPE cells reveals dramatic cellular changes such as an absence of microvilli and vesicular inclusions. The retinoid profile is altered as judged by biochemical analysis, arguing for a partial block in visual pigment regeneration. Surprisingly, homozygous fdv vision mutants survive to adulthood and show, despite a persistence of the hypopigmentation, a partial recovery of retinal morphology. By positional cloning and subsequent morpholino knock-down, we identified a mutation in the silver gene as the molecular defect underlying the fdv phenotype. The Silver protein is required for intralumenal fibril formation in melanosomes by amylogenic cleavage. Our data reveal an unexpected link between melanosome biogenesis and the visual system, undetectable in cell culture.     

Estimating Culicoides sonorensis biting midge abundance using digital image analysis

ImageJ is an open‐source software tool used for a variety of scientific objectives including cell counting, shape analysis and image correction. This technology has previously been used to estimate mosquito abundance in surveillance efforts. However, the utility of this application for estimating abundance or parity in the surveillance of Culicoides spp. (Diptera: Ceratopogonidae) has not yet been tested. Culicoides sonorensis (Wirth and Jones), a biting midge often measuring 2.0–2.5 mm in length, is an economically important vector of ruminant arboviruses in California. Current surveillance methods use visual sorting for the characteristics of midges and are very time‐intensive for large studies. This project tested the utility of ImageJ as a tool to assist in gross trap enumeration as well as in parity analysis of C. sonorensis in comparison with traditional visual methods of enumeration using a dissecting microscope. Results confirmed that automated counting of midges is a reliable means of approximating midge numbers under certain conditions. Further evaluation confirmed accurate and time‐efficient parity analysis in comparison with hand sorting. The ImageJ software shows promise as a tool that can assist and expedite C. sonorensis surveillance. Further, these methods may be useful in other insect surveillance activities.     

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.     

Quality of Visual Cue Affects Visual Reweighting in Quiet Standing

Sensory reweighting is a characteristic of postural control functioning adopted to accommodate environmental changes. The use of mono or binocular cues induces visual reduction/increment of moving room influences on postural sway, suggesting a visual reweighting due to the quality of available sensory cues. Because in our previous study visual conditions were set before each trial, participants could adjust the weight of the different sensory systems in an anticipatory manner based upon the reduction in quality of the visual information. Nevertheless, in daily situations this adjustment is a dynamical process and occurs during ongoing movement. The purpose of this study was to examine the effect of visual transitions in the coupling between visual information and body sway in two different distances from the front wall of a moving room. Eleven young adults stood upright inside of a moving room in two distances (75 and 150 cm) wearing a liquid crystal lenses goggles, which allow individual lenses transition from opaque to transparent and vice-versa. Participants stood still during five minutes for each trial and the lenses status changed every one minute (no vision to binocular vision, no vision to monocular vision, binocular vision to monocular vision, and vice-versa). Results showed that farther distance and monocular vision reduced the effect of visual manipulation on postural sway. The effect of visual transition was condition dependent, with a stronger effect when transitions involved binocular vision than monocular vision. Based upon these results, we conclude that the increased distance from the front wall of the room reduced the effect of visual manipulation on postural sway and that sensory reweighting is stimulus quality dependent, with binocular vision producing a much stronger down/up-weighting than monocular vision.