From microbes to numbers: extracting meaningful quantities from images

Light microscopy offers a unique window into the life and works of microbes and their interactions with hosts. Mere visualization of images, however, does not provide the quantitative information needed to reliably and accurately characterize phenotypes or test computational models of cellular processes, and is unfeasible in high‐throughput screens. Algorithms that automatically extract biologically meaningful quantitative data from images are therefore an increasingly essential complement to the microscopes themselves. This paper reviews some of the computational methods developed to detect, segment and track cells, molecules or viruses, with an emphasis on their underlying assumptions, limitations, and the importance of validation.     

Toward objective selection of representative microscope images

Scientists wishing to communicate the essential characteristics of a pattern (such as an immunofluorescence distribution) currently must make a subjective choice of one or two images to publish. We therefore developed methods for objectively choosing a typical image from a set, with emphasis on images from cell biology. The methods involve calculation of numerical features to describe each image, calculation of similarity between images as a distance in feature space, and ranking of images by distance from the center of the feature distribution. Two types of features were explored, image texture measures and Zernike polynomial moments, and various distance measures were utilized. Criteria for evaluating methods for assigning typicality were proposed and applied to sets of images containing more than one pattern. The results indicate the importance of using distance measures that are insensitive to the presence of outliers. For collections of images of the distributions of a lysosomal protein, a Golgi protein, and nuclear DNA, the images chosen as most typical were in good agreement with the conventional understanding of organelle morphologies. The methods described here have been implemented in a web server (http://murphylab.web.cmu.edu/services/TyplC).     

Virtual microscope interface to high resolution histological images

The Hypertext Atlas of Dermatopathology, the Atlas of Fetal and Neonatal Pathology and Hypertext Atlas of Pathology (this one in Czech only) are available at http://www.muni.cz/atlases. These atlases offer many clinical, macroscopic and microscopic images, together with short introductory texts. Most of the images are annotated and arrows pointing to the important parts of the image can be activated.The Virtual Microscope interface is used for the access to the histological images obtained in high resolution using automated microscope and image stitching, possibly in more focusing planes. Parts of the image prepared in advance are downloaded on demand to save the memory of the user's computer. The virtual microscope is programmed in JavaScript only, works in Firefox/Mozilla and MSIE browsers without need to install any additional software.     

Methodology of protistan discovery: from rRNA detection to quality scanning electron microscope images

Each year, thousands of new protistan 18S rRNA sequences are detected in environmental samples. Many of these sequences are molecular signatures of new protistan species, classes, and/or kingdoms that have never been seen before. The main goal of this study was to enable visualization of these novel organisms and to conduct quality ultrastructural examination. We achieved this goal by modifying standard procedures for cell fixation, fluorescence in situ hybridization, and scanning electron microscopy (SEM) and by making these methodologies work in concert. As a result, the same individual cell can now be detected by 18S rRNA-targeted fluorochrome-labeled probes and then viewed by SEM to reveal its diagnostic morphological characteristics. The method was successfully tested on a wide range of protists (alveolates, stramenopiles, kinetoplastids, and cryptomonads). The new methodology thus opens a way for fine microscopy studies of many organisms previously known exclusively by their 18S rRNA sequences.     

Software for quantification of labeled bacteria from digital microscope images by automated image analysis

Automated image analysis software, CellC, was developed and validated for quantification of bacterial cells from digital microscope images. CellC enables automated enumeration of bacterial cells, comparison of total count and specific count images [e.g., 4',6-diamino-2-phenylindole (DAPI) and fluorescence in situ hybridization (FISH) images], and provides quantitative estimates of cell morphology. The software includes an intuitive graphical user interface that enables easy usage as well as sequential analysis of multiple images without user intervention. Validation of enumeration reveals correlation to be better than 0.98 when total bacterial counts by CellC are compared with manual enumeration, with all validated image types. The software is freely available and modifiable: the executable files and MATLAB source codes can be obtained at www. cs. tut.fi/sgn/csb/cellc.     

Automated recognition of intracellular organelles in confocal microscope images

Recognition of the localisation of intracellular proteins is essential to the understanding of their function. It is usually made through knowledge of and comparison to the distribution of well-characterised intracellular organelles by experts in cell biology. We have automated this process in order to achieve a more objective and quantitative assessment of the protein distribution within the cell, which can be employed by the less experienced cell biologist and may be utilised as a training program for inexperienced users, or as a high throughput localisation program for novel genes in functional analysis. Here we describe the development and testing of a classification system based on a modular neural network trained with sets of confocal sections through cell lines fluorescently stained for markers of key intracellular structures. The system functioned well in spite of the variability in pattern that occurs between individual cells and performed with 97% accuracy, which gives us confidence in the method and in its future development. It is envisaged that this program will aid the design of further experiments utilising colocalisation with known organelle marker proteins, in order to confirm putative trafficking pathways and protein–protein interactions of the protein of interest.     

High resolution electron microscope lattice images of powdered gallstones

Atomic structural images of gallstones were characterized by high-resolution electron microscopy. Regardless of the types of gallstones there was a broad spectrum of crystal size and perfection observed in the stone particles. The crystalline zones in the stone matrix were characterized by well-defined, strongly bounded, ordered arrangement of atoms or atom clusters, while the amorphous zones were characterized by an interlacing network of granulo-fibrillar substructures. The granulo-fibrillar substructures in the amorphous zone could be seen to bond tightly to the lattice fringes in the crystalline zone along the interfaces between two zones. The crystal lattices at the boundary between the crystals were very well matched with the periodicity of atoms continuous through the two crystals. It is hoped that these new electron microscopic data principally related to the structural organization in the gallstones at the atomic or near-atomic level are helpful to a more complete understanding of the micromechanisms of gallstone formation.     

Examining Agreement between Clinicians when Assessing Sick Children

Background

Case management guidelines use a limited set of clinical features to guide assessment and treatment for common childhood diseases in poor countries. Using video records of clinical signs we assessed agreement among experts and assessed whether Kenyan health workers could identify signs defined by expert consensus.

Methodology

104 videos representing 11 clinical sign categories were presented to experts using a web questionnaire. Proportionate agreement and agreement beyond chance were calculated using kappa and the AC1 statistic. 31 videos were selected and presented to local health workers, 20 for which experts had demonstrated clear agreement and 11 for which experts could not demonstrate agreement.

Principal Findings

Experts reached very high level of chance adjusted agreement for some videos while for a few videos no agreement beyond chance was found. Where experts agreed Kenyan hospital staff of all cadres recognised signs with high mean sensitivity and specificity (sensitivity: 0.897–0.975, specificity: 0.813–0.894); years of experience, gender and hospital had no influence on mean sensitivity or specificity. Local health workers did not agree on videos where experts had low or no agreement. Results of different agreement statistics for multiple observers, the AC1 and Fleiss'' kappa, differ across the range of proportionate agreement.

Conclusion

Videos provide a useful means to test agreement amongst geographically diverse groups of health workers. Kenyan health workers are in agreement with experts where clinical signs are clear-cut supporting the potential value of assessment and management guidelines. However, clinical signs are not always clear-cut. Video recordings offer one means to help standardise interpretation of clinical signs.     

Assessing hemorrhagic shock: Feasibility of using an ultracompact photoacoustic microscope

Hemorrhagic shock, as an important clinical issue, is regarding as a critical disease with a high mortality rate. Unfortunately, existing clinical technologies are inaccessible to assess the hemorrhagic shock via hemodynamics in microcirculation. Here, we propose an ultracompact photoacoustic microscope to assess hemorrhagic shock using a rat model and demonstrate its clinical feasibility by visualizing buccal microcirculation of healthy volunteers. Both functional and morphological features of the microvascular network including concentration of total hemoglobin (C_HbT), number of blood vessels (VN), small vascular density (SVD) and vascular diameter (VD) were derived to assess the microvascular hemodynamics of different organs. Animal studies show the feasibility of the proposed tool to assess and stage the hemorrhagic shock via microcirculation. in vivo oral imaging of healthy volunteers indicates the translational possibility of this technique for clinical evaluation of hemorrhagic shock.      

Colocalization of fluorescent markers in confocal microscope images of plant cells

This protocol describes the steps needed to perform quantitative statistical colocalization on two-color confocal images, specifically of plant cells. The procedure includes a calibration test to check the chromatic alignment of the confocal microscope. A software tool is provided to calculate the Pearson and Spearman correlation coefficients ('Pearson-Spearman correlation colocalization' ImageJ plug-in) across regions of interest within the image. Steps are included to help the user practice using the software. The result is a quantitative estimate of the amount of colocalization in the images. Manual masking takes about 1-15 min per image, depending on the detail required, and calculating the correlation coefficients is almost instantaneous. Examples of suitable dyes for such two-color colocalization include Oregon Green or Alexa Fluor 488 dyes in the green range (excited with 488-nm laser line) and Alexa Fluor 555 dye in the red range (excited with 543-nm laser line).     

Interactive visual exploration of overlapping similar structures for three-dimensional microscope images

Background

Recent advances in microscopy enable the acquisition of large numbers of tomographic images from living tissues. Three-dimensional microscope images are often displayed with volume rendering by adjusting the transfer functions. However, because the emissions from fluorescent materials and the optical properties based on point spread functions affect the imaging results, the intensity value can differ locally, even in the same structure. Further, images obtained from brain tissues contain a variety of neural structures such as dendrites and axons with complex crossings and overlapping linear structures. In these cases, the transfer functions previously used fail to optimize image generation, making it difficult to explore the connectivity of these tissues.

Results

This paper proposes an interactive visual exploration method by which the transfer functions are modified locally and interactively based on multidimensional features in the images. A direct editing interface is also provided to specify both the target region and structures with characteristic features, where all manual operations can be performed on the rendered image. This method is demonstrated using two-photon microscope images acquired from living mice, and is shown to be an effective method for interactive visual exploration of overlapping similar structures.

Conclusions

An interactive visualization method was introduced for local improvement of visualization by volume rendering in two-photon microscope images containing regions in which linear nerve structures crisscross in a complex manner. The proposed method is characterized by the localized multidimensional transfer function and interface where the parameters can be determined by the user to suit their particular visualization requirements.     

High-resolution electron microscope and computed images of human tooth enamel crystals

The structure of human enamel crystallites has been studied at a near atomic level by high-resolution electron microscopy. Electron micrographs have been obtained from crystallites present in human enamel with a structure resolution of 0.2 nm in the [0001], [1210], [1213], [1100] and [4510] zone axes directions. In most cases it was possible to match the experimental images with images calculated using the atomic positions of mineral hydroxyapatite. However, in some cases a discrepancy between calculated and experimental image detail was observed in the c direction of the [1210] and the [1100] images. This shows: (i) a structural heterogeneity of the crystals, and (ii) a loss of hexagonal symmetry of the structure. The resolution required to distinguish individual atomic sites in the different zones has been determined, and this will provide a useful basis for future work. As the determination of the "real structure" of biological crystals is of prime importance for the study of calcification mechanisms (crystal growth), biological properties and destructive phenomena of calcified tissues (i.e., dental caries and bone resorption).     

Assessing and managing risk when sharing aggregate genetic variant data

Access to genetic data across studies is an important aspect of identifying new genetic associations through genome-wide association studies (GWASs). Meta-analysis across multiple GWASs with combined cohort sizes of tens of thousands of individuals often uncovers many more genome-wide associated loci than the original individual studies; this emphasizes the importance of tools and mechanisms for data sharing. However, even sharing summary-level data, such as allele frequencies, inherently carries some degree of privacy risk to study participants. Here we discuss mechanisms and resources for sharing data from GWASs, particularly focusing on approaches for assessing and quantifying the privacy risks to participants that result from the sharing of summary-level data.