Gebiss: an ImageJ plugin for the specification of ground truth and the performance evaluation of 3D segmentation algorithms.

Background: Image segmentation is a crucial step in quantitative microscopy that helps to define regions of tissues, cells or subcellular compartments. Depending on the degree of user interactions, segmentation methods can be divided into manual, automated or semi-automated approaches. 3D image stacks usually require automated methods due to their large number of optical sections. However, certain applications benefit from manual or semi-automated approaches. Scenarios include the quantification of 3D images with poor signal-to-noise ratios or the generation of so-called ground truth segmentations that are used to evaluate the accuracy of automated segmentation methods. Results: We have developed Gebiss; an ImageJ plugin for the interactive segmentation, visualisation and quantification of 3D microscopic image stacks. We integrated a variety of existing plugins for threshold-based segmentation and volume visualisation. Conclusions: We demonstrate the application of Gebiss to the segmentation of nuclei in live Drosophila embryos and the quantification of neurodegeneration in Drosophila larval brains. Gebiss was developed as a cross-platform ImageJ plugin and is freely available on the web at http://imaging.bii.a-star.edu.sg/projects/gebiss

An automated pattern recognition system for classifying indirect immunofluorescence images for HEp-2 cells and specimens

AbstractImmunofluorescence antinuclear antibody tests are important for diagnosis and management of autoimmune conditions; a key step that would benefit from reliable automation is the recognition of subcellular patterns suggestive of different diseases. We present a system to recognize such patterns, at cellular and specimen levels, in images of HEp-2 cells. Ensembles of SVMs were trained to classify cells into six classes based on sparse encoding of texture features with cell pyramids, capturing spatial, multi-scale structure. A similar approach was used to classify specimens into seven classes. Software implementations were submitted to an international contest hosted by ICPR 2014 (Performance Evaluation of Indirect Immunofluorescence Image Analysis Systems). Mean class accuracies obtained on heldout test data sets were 87.1% and 88.5% for cell and specimen classification respectively. These were the highest achieved in the competition, suggesting that our methods are state-of-the-art. We provide detailed descriptions and extensive experiments with various features and encoding methods

Quantitative Imaging in Electron and Confocal Microscopies for Applications in Biology

Among the large number of topics related to the quantification of images in electron and confocal microscopies for applications in biology, we selected four subjects that we consider to be representative of some recent tendencies. The first is the quantification of three-dimensional data sets recorded routinely in scanning confocal microscopy. The second is the quantification of the textural and fractal appearance of images. The two other topics are related to image series, which are more and more often provided by imaging instruments. The first kind of series concerns electron energy-filtered images. We show that the parametric (modelling) approach can be complemented by non-parametric approaches (e.g., different variants of multivariate statistical techniques). The other kind of series consists of multiple mappings of a specimen. We describe several new tools for the study and quantification of the co-location, with potential application to multiple mappings in microanalysis or in fluorescence microscopy

Reticulon Homology Domain Containing Protein Families of the Endoplasmic Reticulum

The endoplasmic reticulum (ER) is the largest membrane bound organelle in a cell and has multiple responsibilities. Execution of the various duties performed by the ER requires it to be shaped in a rather complex and intricate manner. ER’s two major structural motives, namely sheets and tubules, play very complex yet not fully understood role in giving ER its overall structure and function. The ratio of sheet and tubule conformations differ significantly within cell types and during cell cycle. Such a balance is possible only with a well-functioning set of factors that constantly communicate with each other throughout a cell cycle. These factors are specifically responsible for either shaping the ER sheets or tubules in addition to factors that keep the dynamic nature of the ER sound. During mitosis, ER undergoes a major transformation in its structure, where the sheet-tubule ratio shifts more towards tubules. Specific factors keep this process sound by acting actively during the stage of mitosis for proper cell division to occur. Although research on such factors are still on-going, many in-depth details on such factors (e.g. their precise localization) and their mechanism of action plus novel factors for ER shaping still needs to be resolved using techniques involving high end light and electron microscopy. In addition, a constant battle in data analysis for answering key questions also persists. Development of tools to study and analyse data on the lines of image analysis and processing is an unmet need that needs simultaneous attention. The research in this thesis focuses on three family of proteins that we uncover as responsible candidates in shaping the ER. To aid the study, this thesis also discusses the development of a software platform for analysis of microscopic data generated during this study. In this research, Reticulon family of proteins (RTN) were characterised using high-end microscopic techniques. We showed RTN4A and RTN4B to localize to ER tubules and sheet edges using pre-embedding immuno electron microscopy (immuno-EM) and electron tomography. Using qPCR, RTN4A and RTN4B were observed to be the most expressed isoforms in neurons and epithelial cells respectively. FAM134C, a poorly characterised protein was identified as one of the RTN4B interacting proteins. FAM134C localised to the ER where it specifically resided at high curvature ER (sheet edges and tubules) similar to RTN4B. FAM134C, similar to the RTN4B also had the capability to promote ER tubules upon overexpression. In addition, another family of proteins belonging to receptor expression enhancing protein (REEP), namely REEP3 and REEP4 were studied for shaping ER during mitotic stage of cell cycle. REEP3 and REEP4 collectively were observed both in tubulating peripheral ER during mitosis and clearing tubular ER from the chromatin for a normal mitosis to take place. Collectively, this work elaborates on proteins belonging to three classes that shape and position the ER specifically either in interphase or during stages of cell division. Our findings also throws light on the role of different domains in each of these proteins such as the reticulon homology domain (RHD) that was observed to be present in all these proteins under study. The RHD previously known for inserting partially and unsymmetrically in the outer leaflet of the ER gives a strong indication for proteins like RTN4B and FAM134C to localize to ER thus tubulating ER upon overexpression conditions. We uncovered the RHD’s crucial role in ER shaping and positioning in REEP3/4 during mitosis.Endoplasma- eli solulimakalvosto (engl. endoplasmic reticulum, ER) on solun suurin kalvon rajoittama organelli, ja sillä on useita tehtäviä. Pystyäkseen suoriutumaan eri tehtävistään ER:n rakenne on monimuotoinen ja alati muuntautuva. ER:n päärakenteet, laatat ja tubulukset, muodostavat monimutkaisen verkoston, eikä niiden kaikkia toimintoja täysin vielä tunneta. Laattojen ja tubulusten määrällinen suhde on erilainen eri solutyypeissä ja solusyklin vaiheissa. ER:n toimivan tasapainon saavuttamiseksi tarvitaan useita tekijöitä, jotka ovat vuorovaikutuksessa keskenään koko solusyklin ajan. Osa näistä tekijöistä osallistuu ER:n rakenteen muokkaamiseen ja osa on vastuussa ER:n dynaamisesta luonteesta. Solunjakautumisen aikana ER:n rakenne muuttuu, ja tubulaarisia rakenteita muodostuu suhteellisesti enemmän. Eri tekijät toimivat aktiivisesti solunjakautumisen eri vaiheissa mahdollistaen näin solujen jakautumisen. Nämä tekijät ovat edelleen tutkimuksen kohteena, ja yksityiskohtien esim. tarkan paikantumisen ja toimintamekanismin selvittämiseksi sekä vielä tuntemattomien, ER:n rakenteeseen vaikuttavien tekijöiden löytämiseksi, on käytettävä kehittyneitä tekniikoita kuten valo- ja elektronimikroskopiaa. Myös tietoaineiston analysoinnin täytyy edelleen kehittyä pystyäksemme vastaamaan tärkeisiin kysymyksiin, ja sen vuoksi sekä kuvankäsittelyyn että kuvien analysointiin tarvittavien ohjelmien kehittämiseen on kiinnitettävä erityistä huomiota. Tässä väitöskirjatutkimuksessa tutkittiin kolmea proteiiniperhettä, joiden osoitettiin vaikuttavan ER:n rakenteeseen. Tutkimuksen aikana otettujen mikroskooppikuvien analysointi oli tämän tutkimuksen kannalta oleellista ja tästä johtuen työssä käsitellään myös kuvankäsittelyohjelmiston kehittämistä. Tässä väitöskirjatutkimuksessa karakterisoitiin Reticulon-proteiineja (RTN) uusilla, kehittyneillä mikroskooppisilla tekniikoilla. Immuunielektronimikroskopialla ja elektronitomografialla osoitettiin RTN4A:n ja RTN4B:n paikallistuvan ER:n tubuluksiin ja laattojen reunoille. Kvantitatiivisella polymeraasiketjureaktiolla pystyttiin osoittamaan, että RTN4A on eniten ilmennetty muunnos hermosoluissa ja RTN4B vastaavasti pintakudossoluissa. Vähän tutkittu proteiini, FAM134C, tunnistettiin yhdeksi RTN4B:n kanssa vuorovaikutuksessa olevista proteiineista ja se paikallistettiin samankaltaisiin ER:n rakenteisiin kuin RTN4B (laattojen reunat ja tubulukset). FAM134C:n ja RTN4B:n ylituotto aikaansai tubulaaristen rakenteiden muodostamista. Lisäksi, ER:n rakenneproteiiniryhmän REEP (engl. receptor expression enhancing protein) proteiinien REEP3 ja REEP4 vaikutusta ER:n rakenteeseen tutkittiin solunjakautumisvaiheen aikana. Solunjakautumisvaiheessa REEP3 ja REEP4 paikallistettiin tubulaariseen, perifeeraaliseen ER:ään. Nämä proteiinit tarvittiin myös tubulaarisen ER:n irrottamiseksi kromatiinista normaalin solunjakautumisen aikaansaamiseksi. Tämä tutkimus syventää tietoja kolmen ER:ä muokkaavan proteiiniperheen proteiineista ja niiden paikallistumisesta ja vaikutuksista ER:n rakenteeseen niin kasvuvaiheessa kuin solunjakautumisen eri vaiheissa. Tulokset antavat myös lisätietoa eri domeenien rooleista näissä proteiineissa, esim. retikulonihomologia-domeenista (RHD), jonka löydettiin kaikista näistä proteiineista. RHD:n tiedetään olevan osittain kaksoiskalvorakenteen sisällä ja siten aiheuttavan kalvojen kaarevoitumista: tämä havaittiin myös ER:n tubuloitumisena ylituotettaessa RTN4B:tä tai FAM134C:tä. Tutkimuksen mukaan RHD:lla oli ratkaiseva rooli ER:n REEP 3/4 rakenneproteiinien toiminnassa solunjakautumisen aikana

COMPREHENSIVE PERFORMANCE EVALUATION AND OPTIMIZATION OF HIGH THROUGHPUT SCANNING MICROSCOPY FOR METAPHASE CHROMOSOME IMAGING

Specimen scanning is a critically important tool for diagnosing the genetic diseases in today’s hospital. In order to reduce the clinician’s work load, many investigations have been conducted on developing automatic sample screening techniques in the last twenty years. However, the currently commercialized scanners can only accomplish the low magnification sample screening (i.e. under 10× objective lens), and still require clinicians’ manual operation for the high magnification image acquisition and confirmation (i.e. under 100× objective lens). Therefore, a new high throughput scanning method is recently proposed to continuously scan the specimen and select the clinically analyzable cells. In the medical imaging lab, University of Oklahoma, a prototype of high throughput scanning microscopy is built based on the time delay integration (TDI) line scanning detector. This new scanning method, however, raises several technical challenges for evaluating and optimizing the performance. First, we need to use the clinical samples to compare this new prototype with the conventional two-step scanners. Second, the system DOF should be investigated to assess the impact on clinically analyzable metaphase chromosomes. Further, in order to achieve the optimal results, we should carefully assess and select the auto-focusing methods for the high throughput scanning system. Third, we need to optimize the scanning scheme by finding the optimal trade-off between the image quality and efficiency. Finally, analyzing the performance of the various image features is meaningful for improving the performance of the computer aided detection (CAD) scheme under the high throughput scanning condition. The purpose of this dissertation is to comprehensively evaluate the performance of the high throughput scanning prototype. The first technical challenge was solved by the first investigation, which utilized a number of 9 slides from five patients to compare the detecting performance of the high throughput scanning prototype. The second and third studies were performed for the second technical challenge. In the second study, we first theoretically computed the DOF of our prototype and then experimentally measured the system DOF. After that, the DOF impact was analyzed using cytogenetic images from different pathological specimens, under the condition of two objective lenses of 60× (dry, N.A. = 0.95) and 100× (oil, N.A. = 1.25). In the third study, five auto-focusing functions were investigated using metaphase chromosome images. The performance of these different functions was compared using four widely accepted criteria. The fourth and fifth investigations were designed for the third technical challenge. The fourth study objectively assessed chromosome band sharpness by a gradient sharpness function. The sharpness of the images captured from standard resolution target and several pathological chromosomes was objectively evaluated by the gradient sharpness function. The fifth study presented a new slide scanning scheme, which only applies the auto-focusing operations on limited locations. The focusing position was adjusted very quickly by linear interpolation for the other locations. The sixth study was aimed for the fourth technical challenge. The study investigated 9 different feature extraction methods for the CAD modules applied on our high throughput scanning prototype. A certain amount of images were first acquired from 200 bone marrow cells. Then the tested features were performed on these images and the images containing clinically meaningful chromosomes were selected using each feature individually. The identifying accuracy of each feature was evaluated using the receiver operating characteristic (ROC) method. In this dissertation, we have the following results. First, in most cases, we demonstrated that the high throughput scanning can select more diagnostic images depicting clinically analyzable metaphase chromosomes. These selected images were acquired with adequate spatial resolution for the following clinical interpretation. Second, our results showed that, for the commonly used pathological specimens, the metaphase chromosome band patterns are clinically recognizable when these chromosomes were obtained within 1.5 or 1.0 μm away from the focal plane, under the condition of applying the two 60× or 100× objective lenses, respectively. In addition, when scanning bone marrow and blood samples, the Brenner gradient and threshold pixel counting methods can achieve the optimal performance, respectively. Third, we illustrated that the optimal scanning speed of clinical samples is 0.8 mm/s, for which the captured image sharpness is optimized. When scanning the blood sample slide with an auto-focusing distance of 6.9 mm, the prototype obtained an adequate number of analyzable metaphase cells. More useful cells can be captured by increasing the auto-focusing operations, which may be needed for the high accuracy diagnosis. Finally, we found that the optimal feature for the online CAD scheme is the number of the labeled regions. When applying the offline CAD scheme, the satisfactory results can be achieved by combining four different features including the number of the labeled regions, average region area, average region pixel value, and the standard deviation of the either region circularity or distance. Although these investigations are encouraging, there exist several limitations. First, the number of the specimens is limited in most of the assessments. Second, some important impacts, such as the DOF of human eye and the sample thickness, are not considered. Third, more recently proposed algorithms and image features are not used for the evaluation. Therefore, several further studies are planned, which may provide more meaningful information for improving the scanning efficiency and image quality. In summary, we believe that the high throughput scanning may be extensively applied for diagnosing genetic diseases in the future

Fusion genes in breast cancer

Fusion genes caused by chromosomal rearrangements are a common and important feature in haematological malignancies, but have until recently been seen as unimportant in epithelial cancers. The discovery of recurrent fusion genes in prostate and lung cancer suggests that fusion genes may play an important role in epithelial carcinogenesis, and that they have been previously under-reported due to the difficulties of cytogenetic analysis of solid tumours. In particular, breast cancers often have complex, highly rearranged karyotypes which have proved difficult to analyse using classical cytogenetic techniques. The aim of this project was to search for fusion genes in breast cancer by using high-resolution mapping of chromosome rearrangements in breast cancer cell lines. Mapping the chromosome rearrangements was initially done using high-resolution DNA microarrays and fluorescence in- situ hybridisation, but moved to high-throughput sequencing as it became available. Interesting candidate genes identified from the mapped chromosome rearrangements were investigated on a larger set of cell lines and primary tumours. The complete karyotypes of two breast cancer cell lines were constructed using a combination of microarrays, fluorescence microscopy, and high-throughput sequencing. A number of potential fusion genes were identified in these two cell lines. Although no expressed fusion genes were found, the complete karyotypes gave insight into the number and mechanisms of chromosome rearrangement in breast cancer, and identified interesting candidate genes which may be of importance in tumourigenesis. Two genes which were fused in other breast cancer cell lines, BCAS3 and ODZ4, were disrupted by chromosome rearrangements and identified as interesting candidate genes in tumorigenesis. A bioinformatic pipeline to process high-throughput sequencing data was set up and validated, and shown to more accurately predict fusion genes than other methods, and can be used to investigate further cell lines and tumours for recurrent fusion genes. The pipeline was used to analyse data from 3 other breast cancer cell lines and predict chromosomal rearrangements and fusion genes, several of which were found to be expressed. Of the fusions predicted in the cell line ZR-75-30, 7 expressed fusion genes were identified, and may have functional significance in breast cancer.This work was supported by a grant from Breast Cancer Campaign

Classification and clustering: models, software and applications

We are pleased to present the report on the 30th Fall Meeting of the working group ``Data Analysis and Numerical Classification'' (AG-DANK) of the German Classification Society. The meeting took place at the Weierstrass Institute for Applied Analysis and Stochastics (WIAS), Berlin, from Friday Nov. 14 till Saturday Nov. 15, 2008. Already 12 years ago, WIAS had hosted a traditional Fall Meeting with special focus on classification and multivariate graphics (Mucha and Bock, 1996). This time, the special topics were stability of clustering and classification, mixture decomposition, visualization, and statistical software