Quantifying the branching frequency of virtual filamentous microbes using fractal analysis

The productivity of an industrial fermentation process involving a filamentous microbe is heavily dependent on the morphological form adopted by the organism. The development of systems capable of rapidly and accurately characterizing morphology within a given process represents a significant challenge, as the complex phenotypes that are manifested are not easily quantified. Conventional parameters employed in these analyses are often of limited value, as they reveal little about the branching behavior of the organism; an important consideration given the demonstrated link between branching frequency and metabolite production. In this study, the influence of branching behavior on the spatial distribution of mycelia grown in silico is examined through fractal analysis. It is demonstrated that fractal dimension, quantified based on the frequency distribution of parameterized boundary curves, and lacunarity act as robust estimators of branching behavior. The analysis can, in theory, be applied to any morphological form, providing universally applicable process parameters for more complete data acquisition. Biotechnol. Bioeng. 2013; 110: 437–447. © 2012 Wiley Periodicals, Inc.     

Specimen preparation and quantification of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy

To establish an optimal method for analysis of the collagen structures from unstained tissue sections, a computerized image analysis system using a charge coupled device camera coupled to a polarizing light microscope was used. Retardation values of birefringence, which are proportional to the content and fibril orientation of collagen in the extracellular matrix of articular cartilage, were determined from sections prepared in different ways. In the superficial zone of articular cartilage, the highest retardation values were recorded from sections cut parallel to the so-called split lines indicating the anisotropic arrangement of collagen. Complete digestion of glycosaminoglycans reduced the retardation value by approximately 6.0%, suggesting a minor, but not insignificant, contribution of glycosaminoglycans to the birefringence of the matrix. The use of a mounting medium with a refractive index close to that of the collagen (e.g. DPX) increased the specificity of the method, since the optical anisotropy of collagen derives predominantly from the intrinsic (structural) birefringence. In conclusion, analysis of unstained sections after careful removal of paraffin and glycosaminoglycans from the tissues provides a sensitive and rapid quantitative assessment of oriented collagen structures in articular cartilage     

Colour model analysis for microscopic image processing

This article presents a comparative study between different colour models (RGB, HSI and CIEL*a*b*) applied to a very large microscopic image analysis. Such analysis of different colour models is needed in order to carry out a successful detection and therefore a classification of different regions of interest (ROIs) within the image. This, in turn, allows both distinguishing possible ROIs and retrieving their proper colour for further ROI analysis. This analysis is not commonly done in many biomedical applications that deal with colour images. Other important aspects is the computational cost of the different processing algorithms according to the colour model. This work takes these aspects into consideration to choose the best colour model tailored to the microscopic stain and tissue type under consideration and to obtain a successful processing of the histological image.     

Classification and measurement of fungal pellets by automated image analysis

An automated image analysis method for classifying and measuring pellets of filamentous fungi growing in submerged fermentations has been developed. The method discriminates between pelleted mycelial growth and loose aggregates of dispersed hyphae. Pellets are classified into smooth and hairy types. In both cases, the core of the pellet is identified and its shape and size characterized. For hairy pellets the annular region is also characterized. The method was tested on pellets of Aspergillus niger ATCC 11414 grown in a defined medium in shake flasks. This rapid method makes practical extensive studies on the morphology of pellets in submerged fermentations and the influence of fermentation conditions on that morphology.     

Three-dimensional image processing for morphometric analysis of epithelium sections.

The reproducible classification of poorly differentiated abnormal epithelium specimens is still a diagnostic problem. The computer-aided method described here improves the differentiation between benign and malignant epithelium specimens. Hematoxylin and eosin-stained sections of normal squamous epithelium, dysplasia, carcinoma in situ, and carcinoma were scanned in a TV microscope system and analyzed by means of image processing methods on a DEC 5000/200 workstation. From the 15-20 microns thick histological sections, 3-5 focus positions in steps of 1-4 microns were scanned. The segmentation of the cell nuclei was performed automatically by color analysis and geometric operations. For each nucleus the best focus level was selected and at this level the center of the cell was calculated. Graph theoretical methods were applied to analyze the morphometry of the epithelium specimens. The minimal spanning tree was computed in the three-dimensional (3D) space of the sections with the selected centers of the nuclei as vertices. The best feature found for discrimination of the specimens is the average length of all edges in a tree. In the two-dimensional (2D) analysis we had to accept an error probability of about 20% in differentiation of dysplasia and carcinoma. In contrast to this we differentiated normal squamous epithelium, dysplasia, and carcinoma with a correct classification rate of 100% in the 3D analysis.     

Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing.

BACKGROUND: Clonogenic assays are used frequently to measure the cell killing and mutagenic effects of radiation and other agents. Clonogenic assays carried out manually are tedious and time-consuming and involve a significant element of subjectivity. However, several commercial automatic colony counters are available. Based on CCD video imaging and image analysis they are relatively expensive and can analyze only one petri dish at a time. METHOD: We have developed a cheaper and more efficient device, which employs a flat bed scanner to image 12 60-mm petri dishes at a time. Two major problems in automated colony counting are the clustering of colonies and edge effects. By using standard image analysis and implementing an inflection point algorithm, these problems were greatly diminished. The resulting system was compared with two manual colony counts, as well as with automated counts with the Oxford Optronix ColCount colony counter for cell lines V79 and HaCaT. RESULTS: Comparisons assuming the manual counts to be correct showed that our automatic counter was slightly more accurate than the commercial unit. CONCLUSIONS: As a whole, our automated colony counter performed significantly better than the commercial unit with regard to processing time, cost and accuracy.     

Automatic quantitation of cell colonies on petri dishes by computerized image analysis

Clonogenic assay is one of the most sensitive assays, widely used to evaluate the effects of antineoplastic agentsin vitro. A computer program was developed on an IBAS 2.0 Image Analysis System for automated quantiation of cell colonies and clone area on Petri dishes. The sensitivity of the clonogenic assay can be greatly increased by evaluating the mean area of the clones. The program gives an objective, accurate and fast evaluation of large samples. It is simple to use and offers a high degree of flexibility. Special algorithms and techniques have been implemented for good quantitation of both connected and well-separated colonies and to reduce the background noise and the general error rate. The principles and solutions presented are applicable to any other image analysis system.Abbreviations FBS fetal bovine serum - ATCC American Type Culture Collection - DDATHF 5,10-dideazatetrahydrofolic acid - PBS phosphate buffered saline     

Video image processing greatly enhances contrast, quality, and speed in polarization-based microscopy

Video cameras with contrast and black level controls can yield polarized light and differential interference contrast microscope images with unprecedented image quality, resolution, and recording speed. The theoretical basis and practical aspects of video polarization and differential interference contrast microscopy are discussed and several applications in cell biology are illustrated. These include: birefringence of cortical structures and beating cilia in Stentor, birefringence of rotating flagella on a single bacterium, growth and morphogenesis of echinoderm skeletal spicules in culture, ciliary and electrical activity in a balancing organ of a nudibranch snail, and acrosomal reaction in activated sperm.     

Proximal root diameter as predictor of total root size for fractal branching models

In a fractal branching pattern the same rules govern branching at each subsequent level. The initial size (diameter) and the essential branching rules thus contain the information required to construct the whole pattern. If root branching patterns have fractal characteristics, measurement of the proximal root diameter at the stem base and the branching rules as observed anywhere in the root system, would be enough to predict total root length, root diameter distribution and root length per unit dry weight (specific root length). A ‘pipe stem’ model is used to derive algebraic relations between total root size and proximal root diameter for two classes of branching patterns, determinate and proportionate. To predict total root length from the proximal root diameter, at least information is needed on the minimum root diameter, the average length of internal and external links (segments) and the proportionality factor between total cross sectional areas before and after branching. For the length of the longest root or the specific root length further information on the branching rules is needed, as it is highest for determinate and proportionate branching rules, respectively.     

Labolmage: a workstation environment for research in image processing and analysis

Numerous images are produced daily in biomedical research. Inorder to extract relevant and useful results, various processingand analysis steps are mandatory. The present paper describesa new, powerful and user-friendly image analysis system: Labolmage.In addition to standard image processing modules, Labolmagealso contains various specialized tools. These multiple processingmodules and tools are first introduced. A one-dimensional gelanalysis method is then described. The new concept of ‘normalizedvirtual one-dimensional gel’ is introduced, making comparisonsbetween gels particularly easy. This normalized gel is obtainedby compensating for the bending of the lanes automatically;no information loss is incurred in the process. Finally, themodel of interaction in a multi-window environment is discussed.Labolmage is designed to run in two ways: interactively, usingmenus and panels; and in batch mode by means of user-definedmacros. Examples are given to illustrate the potentialitiesof the software. Received on August 27, 1990; accepted on November 15, 1990     

A rapid quantitative measurement of root length and root branching by microcomputer image analysis

A computer program was made for fast and reliable measurement of root length and for estimating the number of root tips and branching points. Image-processing procedures available in a program package for image analysis by means of a personal computer were used. The method is described in this paper and some results of tests on variance and systematic errors (bias) are discussed.Time required for analysis of an evenly spread root (sub-)sample with a total length of max. 300 cm was reduced to less than 20 seconds. Random deviations from the real length, determined by measuring known lengths of wire, did not exceed 5%, after correction for length density dependent bias. Counts of root tips appeared to be unreliable, but branching ratios could be determined fairly accurately, after correction for the length density dependent number of pseudo-branches (e.g. crossings). Rhizotron root photographs were also analysed satisfactorily, after modification of a few steps in the program.     

Fast preparation of fungal DNA for PCR screening

Rapid DNA preparation for the quick screening is highly demanded in diverse research fields. Here, we combined an extraction buffer and heat treatment to generate DNA templates from yeast and filamentous fungal materials for PCR. This method may be widely applicable to diverse fungal species in clinical and basic studies.     

Comparative analysis of fungal communities in colonies of two leaf-cutting ant species with different substratum preferences

Fungus gardens of leaf-cutting ants harbor diverse alien fungi in addition to their fungal cultivar. Previous work suggested that alien microorganisms are likely derived from the substrata foraged by ant workers and incorporated into the fungus gardens. To test this hypothesis, we sampled 1014 garden fragments from 16 field colonies of Atta sexdens rubropilosa (a dicot-cutting ant) and Atta capiguara (a grass-cutting ant) in Brazil. From a total of 615 fungal isolates recovered, we observed similar diversity of fungi between colonies of both ant species. However, fungal communities differed in composition of taxa between ant colonies. Trichoderma spirale, Trichosporon chiarellii and Penicillium citrinum were prevalent accounting for 18.5%, 12.2% and 11.7% of the total isolates, respectively. As expected, fungal communities clustered in two major groups supporting the hypothesis that plant substratum has an impact on the composition of the alien fungi found in leaf-cutting ant gardens.     

Combination system in advanced image processing for improving image contrast and a conventional row-ratio grid for an indirect flat-panel detector system: An experimental study

Conventional grids with high grid ratios are not ideal for use in bedside radiography because of the difficulty in maintaining the required alignment. To address this issue, the potential usefulness of a combination system that employs removal processing software for scattered radiation and a conventional grid with a low grid ratio (3:1) for an indirect-conversion-type flat-panel detector system was evaluated by measuring image quality and observer performance. The hypothetical grid ratios for the software were 2:1, 3:1, 6:1, 8:1, and 10:1. The scatter fraction of the combination system was lower than that of the software alone. Significant improvement was observed in the effect of scattered radiation removal up to a hypothetical software grid ratio of 6:1. However, the Wiener spectrum increased (radiographic noise degraded) with an increase in the hypothetical grid ratio. The contrast ratios of the combination system were improved compared to those of the software alone for anthropomorphic chest radiographs. An observer test was also conducted using the contrast-detail phantom. The combination system indicated higher low-contrast detectability compared to the software alone, although there were no statistical differences between the hypothetical grid ratios of 6:1, 8:1, and 10:1 in all combinations of the software alone and the combination system. We concluded that a combination system with software that uses a hypothetical grid ratio of 6:1 or more and a 3:1 conventional grid would be more useful for reducing the scattered radiation component compared to the software alone with a hypothetical higher grid ratio for thicker objects.     

Viability testing and characterization of germination of fungal spores by automatic image analysis

Fungal spores are used in the laboratory for culture maintenance and at laboratory and other scales as inocula for fermentations. The spore swelling and germination processes constitute a major part of the lag phase, and the subsequent culture morphology and productivity can be greatly influenced by the initial concentration and condition of the spores. An image analysis method has been developed for assessing the viability and the germination characteristics of fungal spores in submerged cultures. Structural variations during germination, i.e., swelling, germ tube formation, and germ tube elongation, are measured in terms of distributions of spore volumes and of germ tube lengths and volumes. These measurements are fully automatic and give a very rapid assessment of spore viability. This image analysis method might be used as a tool in culture maintenance and for determining the quality of inocula for fungal fermentations. (c) 1993 John Wiley & Sons, Inc.     

Morphological quantification of filamentous fungal development using membrane immobilization and automatic image analysis

Mycelial morphology is a critically important process property in industrial fermentations of filamentous micro-organisms, as particular phenotypes are associated with maximum productivity. However, the accurate quantification of complex morphologies still represents a significant challenge in elucidating this relationship. A system has been developed for high-resolution characterisation of filamentous fungal growth on a solid substrate, using membrane immobilization and fully-automatic plug-ins developed for the public domain, Java-based, image-processing software, ImageJ. The system has been used to quantify the microscopic development of Aspergillus oryzae on malt agar, by measuring spore projected area and circularity, the total length of a hyphal element, the number of tips per element, and the hyphal growth unit. Two different stages of growth are described, from the swelling of a population of conidiospores up to fully developed, branched hyphae 24 h after inoculation. Spore swelling expressed as an increase in mean equivalent spore diameter was found to be approximately linear with time. Widespread germination of spores was observed by 8 h after inoculation. From approximately 12 h, the number of tips was found to increase exponentially. The specific growth rate of a population of hyphae was calculated as approximately 0.24–0.27 h⁻¹. A wide variation in growth kinetics was found within the population. The robustness of the image-analysis system was verified by testing the effect of small variations in the input data.     

Development of a sample preparation method for fungal proteomics

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.