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A detailed analysis of 3D subcellular signal localization
Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
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2009 (English)In: Cytometry Part A, ISSN 1552-4922, Vol. 75A, no 4, 319-328 p.Article in journal (Refereed) Published
Abstract [en]

Detection and localization of fluorescent signals in relation to other subcellular structures is an important task in various biological studies. Many methods for analysis of fluorescence microscopy image data are limited to 2D. As cells are in fact 3D structures, there is a growing need for robust methods for analysis of 3D data. This article presents an approach for detecting point-like fluorescent signals and analyzing their subnuclear position. Cell nuclei are delineated using marker-controlled (seeded) 3D watershed segmentation. User-defined object and background seeds are given as input, and gradient information defines merging and splitting criteria. Point-like signals are detected using a modified stable wave detector and localized in relation to the nuclear membrane using distance shells. The method was applied to a set of biological data studying the localization of Smad2-Smad4 protein complexes in relation to the nuclear membrane. Smad complexes appear as early as 1 min after stimulation while the highest signal concentration is observed 45 min after stimulation, followed by a concentration decrease. The robust 3D signal detection and concentration measures obtained using the proposed method agree with previous observations while also revealing new information regarding the complex formation.

Place, publisher, year, edition, pages
2009. Vol. 75A, no 4, 319-328 p.
Keyword [en]
3D image analysis, fluorescence signal segmentation, subcellular positioning, Smad detection
National Category
Computer and Information Sciences
Identifiers
URN: urn:nbn:se:uu:diva-98014DOI: 10.1002/cyto.a.20663ISI: 000264513800006OAI: oai:DiVA.org:uu-98014DiVA: diva2:173170
Available from: 2009-02-05 Created: 2009-02-05 Last updated: 2018-01-13Bibliographically approved
In thesis
1. Digital Image Analysis of Cells: Applications in 2D, 3D and Time
Open this publication in new window or tab >>Digital Image Analysis of Cells: Applications in 2D, 3D and Time
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Light microscopes are essential research tools in biology and medicine. Cell and tissue staining methods have improved immensely over the years and microscopes are now equipped with digital image acquisition capabilities. The image data produced require development of specialized analysis methods. This thesis presents digital image analysis methods for cell image data in 2D, 3D and time sequences.

Stem cells have the capability to differentiate into specific cell types. The mechanism behind differentiation can be studied by tracking cells over time. This thesis presents a combined segmentation and tracking algorithm for time sequence images of neural stem cells.The method handles splitting and merging of cells and the results are similar to those achieved by manual tracking.

Methods for detecting and localizing signals from fluorescence stained biomolecules are essential when studying how they function and interact. A study of Smad proteins, that serve as transcription factors by forming complexes and enter the cell nucleus, is included in the thesis. Confocal microscopy images of cell nuclei are delineated using gradient information, and Smad complexes are localized using a novel method for 3D signal detection. Thus, the localization of Smad complexes in relation to the nuclear membrane can be analyzed. A detailed comparison between the proposed and previous methods for detection of point-source signals is presented, showing that the proposed method has better resolving power and is more robust to noise.

In this thesis, it is also shown how cell confluence can be measured by classification of wavelet based texture features. Monitoring cell confluence is valuable for optimization of cell culture parameters and cell harvest. The results obtained agree with visual observations and provide an efficient approach to monitor cell confluence and detect necrosis.

Quantitative measurements on cells are important in both cytology and histology. The color provided by Pap (Papanicolaou) staining increases the available image information. The thesis explores different color spaces of Pap smear images from thyroid nodules, with the aim of finding the representation that maximizes detection of malignancies using color information in addition to quantitative morphological parameters.

The presented methods provide useful tools for cell image analysis, but they can of course also be used for other image analysis applications.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2009. 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 596
Keyword
Digital image analysis, microscopy, fluorescent staining, watershed segmentation, sub-cellular localization, point-like signals, wavelets, cell confluence, cytology, color spaces.
National Category
Computer Vision and Robotics (Autonomous Systems)
Identifiers
urn:nbn:se:uu:diva-9541 (URN)978-91-554-7398-3 (ISBN)
Public defence
2009-02-27, Siegbansalen, Ångstrom Laboratory, Polackbacken, Uppsala, 10:15
Opponent
Supervisors
Available from: 2009-02-05 Created: 2009-02-05 Last updated: 2018-01-13Bibliographically approved
2. Methods for 2D and 3D Quantitative Microscopy of Biological Samples
Open this publication in new window or tab >>Methods for 2D and 3D Quantitative Microscopy of Biological Samples
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

New microscopy techniques are continuously developed, resulting in more rapid acquisition of large amounts of data. Manual analysis of such data is extremely time-consuming and many features are difficult to quantify without the aid of a computer. But with automated image analysis biologists can extract quantitative measurements and increases throughput significantly, which becomes particularly important in high-throughput screening (HTS). This thesis addresses automation of traditional analysis of cell data as well as automation of both image capture and analysis in zebrafish high-throughput screening. 

It is common in microscopy images to stain the nuclei in the cells, and to label the DNA and proteins in different ways. Padlock-probing and proximity ligation are highly specific detection methods that  produce point-like signals within the cells. Accurate signal detection and segmentation is often a key step in analysis of these types of images. Cells in a sample will always show some degree of variation in DNA and protein expression and to quantify these variations each cell has to be analyzed individually. This thesis presents development and evaluation of single cell analysis on a range of different types of image data. In addition, we present a novel method for signal detection in three dimensions. 

HTS systems often use a combination of microscopy and image analysis to analyze cell-based samples. However, many diseases and biological pathways can be better studied in whole animals, particularly those that involve organ systems and multi-cellular interactions. The zebrafish is a widely-used vertebrate model of human organ function and development. Our collaborators have developed a high-throughput platform for cellular-resolution in vivo chemical and genetic screens on zebrafish larvae. This thesis presents improvements to the system, including accurate positioning of the fish which incorporates methods for detecting regions of interest, making the system fully automatic. Furthermore, the thesis describes a novel high-throughput tomography system for screening live zebrafish in both fluorescence and bright field microscopy. This 3D imaging approach combined with automatic quantification of morphological changes enables previously intractable high-throughput screening of vertebrate model organisms.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 70 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 856
Keyword
Image analysis, cytmetry, model organism, zebrafish, screening
National Category
Medical Image Processing
Research subject
Computerized Image Processing
Identifiers
urn:nbn:se:uu:diva-159196 (URN)978-91-554-8167-4 (ISBN)
Public defence
2011-11-11, Room 2446, Polacksbacken, Lägerhyddsvägen 2, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2011-10-21 Created: 2011-09-25 Last updated: 2014-07-21Bibliographically approved
3. Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway
Open this publication in new window or tab >>Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A comprehensive understanding of how the body and all its components function is essential when this knowledge is exploited for medical purposes. The achievements in biological and medical research during last decades has provided us with the complete human genome and identified signaling pathways that governs the cellular processes that facilitates the development and maintenance of higher order organisms. This has brought about the realization that diseases such as cancer is a consequence of genomic aberrations that effects these signaling pathways, endowing cancer cells with the capacity to circumvent homeostasis by acquiring features like self-sustained proliferation and insensitivity to apoptosis. The increased understanding of biology and medicine has been made possible by the development of advanced methods to carry out biological and clinical analyses. The demands of a method often differ regarding in what context it will be applied. It may be acceptable for method to be laborious and time consuming if it is used in basic research, but for medical purposes molecular methods need to be fast and straightforward to perform. Innovative technologies should preferentially address the demands of both researchers and clinicians and provide data not possible to obtain by other methods. An example of such a method is the in situ proximity ligation assay (in situ PLA). In this thesis I have used this method to determine the activity status, at the single-cell level, of the transforming growth factor-β (TGF-β) signaling pathway and activating protein-1 (AP-1) family of transcription factors.  Both of these pathways are frequently involved in cancer development and progression. In addition to this research I herein also present further modifications of in situ PLA, and analyses thereof, to increase the utility and resolution of this assay.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 763
Keyword
proximity ligation, TGF-β signaling, Smad, single cell analysis, cell cycle, context-dependent signaling, CRC
National Category
Cell and Molecular Biology Cancer and Oncology Biomedical Laboratory Science/Technology
Research subject
Molecular Cellbiology; Medicinal Chemistry; Molecular Biology
Identifiers
urn:nbn:se:uu:diva-171952 (URN)978-91-554-8336-4 (ISBN)
Public defence
2012-05-25, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-05-02 Created: 2012-03-29 Last updated: 2018-01-12Bibliographically approved

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