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  • 1.
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Methods for 2D and 3D Quantitative Microscopy of Biological Samples2011Doctoral 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.

    List of papers
    1. A detailed analysis of 3D subcellular signal localization
    Open this publication in new window or tab >>A detailed analysis of 3D subcellular signal localization
    Show others...
    2009 (English)In: Cytometry Part A, ISSN 1552-4922, Vol. 75A, no 4, p. 319-328Article 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.

    Keywords
    3D image analysis, fluorescence signal segmentation, subcellular positioning, Smad detection
    National Category
    Computer and Information Sciences
    Identifiers
    urn:nbn:se:uu:diva-98014 (URN)10.1002/cyto.a.20663 (DOI)000264513800006 ()
    Available from: 2009-02-05 Created: 2009-02-05 Last updated: 2018-01-13Bibliographically approved
    2. Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis
    Open this publication in new window or tab >>Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis
    Show others...
    2007 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 55, no 11, p. 1159-1166Article in journal (Refereed) Published
    Abstract [en]

    Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification–based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.

    Keywords
    A3243G mtDNA, Aging, Heteroplasmy, Mitochondrial diseases, Mutation load, Padlock probing, PCR-RFLP, Segregation
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-12658 (URN)10.1369/jhc.7A7282.2007 (DOI)000250320100009 ()17679731 (PubMedID)
    Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2017-12-11Bibliographically approved
    3. BlobFinder, a tool for fluorescence microscopy image cytometry
    Open this publication in new window or tab >>BlobFinder, a tool for fluorescence microscopy image cytometry
    2009 (English)In: Computer Methods and Programs in Biomedicine, ISSN 0169-2607, E-ISSN 1872-7565, Vol. 94, no 1, p. 58-65Article in journal (Refereed) Published
    Abstract [en]

    Images can be acquired at high rates with modern fluorescence microscopy hardware, giving rise to a demand for high-speed analysis of image data. Digital image cytometry, i.e., automated measurements and extraction of quantitative data from images of cells, provides valuable information for many types of biomedical analysis. There exists a number of different image analysis software packages that can be programmed to perform a wide array of useful measurements. However, the multi-application capability often compromises the simplicity of the tool. Also, the gain in speed of analysis is often compromised by time spent learning complicated software. We provide a free software called BlobFinder that is intended for a limited type of application, making it easy to use, easy to learn and optimized for its particular task. BlobFinder can perform batch processing of image data and quantify as well as localize cells and point like source signals in fluorescence microscopy images, e.g., from FISH, in situ PLA and padlock probing, in a fast and easy way.

    Keywords
    Image cytometry, Single cell analysis, FISH, Software
    National Category
    Computer Vision and Robotics (Autonomous Systems)
    Research subject
    Computerized Image Analysis
    Identifiers
    urn:nbn:se:uu:diva-87971 (URN)10.1016/j.cmpb.2008.08.006 (DOI)000264282400006 ()18950895 (PubMedID)
    Available from: 2009-01-22 Created: 2009-01-16 Last updated: 2018-06-26Bibliographically approved
    4. Robust signal detection in 3D fluorescence microscopy
    Open this publication in new window or tab >>Robust signal detection in 3D fluorescence microscopy
    2010 (English)In: Cytometry. Part A, ISSN 1552-4922, Vol. 77A, no 1, p. 86-96Article in journal (Refereed) Published
    Abstract [en]

    Robust detection and localization of biomolecules inside cells is of great importance to better understand the functions related to them. Fluorescence microscopy and specific staining methods make biomolecules appear as point-like signals on image data, often acquired in 3D. Visual detection of such point-like signals can be time consuming and problematic if the 3D images are large, containing many, sometimes overlapping, signals. This sets a demand for robust automated methods for accurate detection of signals in 3D fluorescence microscopy. We propose a new 3D point-source signal detection method that is based on Fourier series. The method consists of two parts, a detector, which is a cosine filter to enhance the point-like signals, and a verifier, which is a sine filter to validate the result from the detector. Compared to conventional methods, our method shows better robustness to noise and good ability to resolve signals that are spatially close. Tests on image data show that the method has equivalent accuracy in signal detection in comparison to Visual detection by experts. The proposed method can be used as an efficient point-like signal detection tool for various types of biological 3D image data.

    National Category
    Bioinformatics and Systems Biology
    Identifiers
    urn:nbn:se:uu:diva-98015 (URN)10.1002/cyto.a.20795 (DOI)000273384700011 ()
    Available from: 2009-02-05 Created: 2009-02-05 Last updated: 2011-11-04Bibliographically approved
    5. High-throughput in vivo optical projection tomography of small vertebrates
    Open this publication in new window or tab >>High-throughput in vivo optical projection tomography of small vertebrates
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-159203 (URN)
    Available from: 2011-09-25 Created: 2011-09-25 Last updated: 2011-11-04
    6. Fully automated cellular-resolution vertebrate screening platform with parallel animal processing
    Open this publication in new window or tab >>Fully automated cellular-resolution vertebrate screening platform with parallel animal processing
    Show others...
    2012 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 12, no 4, p. 711-716Article in journal (Refereed) Published
    Abstract [en]

    The zebrafish larva is an optically-transparent vertebrate model with complex organs that is widelyused to study genetics, developmental biology, and to model various human diseases. In this article, wepresent a set of novel technologies that significantly increase the throughput and capabilities of ourpreviously described vertebrate automated screening technology (VAST). We developed a robustmulti-thread system that can simultaneously process multiple animals. System throughput is limitedonly by the image acquisition speed rather than by the fluidic or mechanical processes. We developedimage recognition algorithms that fully automate manipulation of animals, including orienting andpositioning regions of interest within the microscope’s field of view. We also identified the optimalcapillary materials for high-resolution, distortion-free, low-background imaging of zebrafish larvae.

    National Category
    Computer Vision and Robotics (Autonomous Systems)
    Identifiers
    urn:nbn:se:uu:diva-159202 (URN)10.1039/c1lc20849g (DOI)000299380800007 ()
    Available from: 2011-09-25 Created: 2011-09-25 Last updated: 2018-01-12Bibliographically approved
    7. Image based measurements of single cell mtDNA mutation load MTD 2007
    Open this publication in new window or tab >>Image based measurements of single cell mtDNA mutation load MTD 2007
    Show others...
    2007 (English)In: Medicinteknikdagarna 2007, 2007Conference paper, Published paper (Other (popular science, discussion, etc.))
    Abstract [en]

    Cell cultures as well as cells in tissue always display a certain degree of variability,and measurements based on cell averages will miss important information contained in a heterogeneous population. These differences among cells in a population may be essential to quantify when looking at, e.g., protein expression and mutations in tumor cells which often show high degree of heterogeneity.

    Single nucleotide mutations in the mithochondrial DNA (mtDNA) can accumulate and later be present in large proportions of the mithocondria causing devastating diseases. To study mtDNA accumulation and segregation one needs to measure the amount of mtDNA mutations in each cell in multiple serial cell culture passages. The different degrees of mutation in a cell culture can be quantified by making measurements on individual cells as an alternative to looking at an average of a population. Fluorescence microscopy in combination with automated digital image analysis provides an efficient approach to this type of single cell analysis.

    Image analysis software for these types of applications are often complicated and not easy to use for persons lacking extensive knowledge in image analysis, e.g., laboratory personnel. This paper presents a user friendly implementation of an automated method for image based measurements of mtDNA mutations in individual cells detected with padlock probes and rolling-circle amplification (RCA). The mitochondria are present in the cell’s cytoplasm, and here each cytoplasm has to be delineated without the presence of a cytoplasmic stain. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%. The final image based measurements of mitochondrial mutation load are also compared to, and show high agreement with, measurements made using biochemical techniques.

    National Category
    Other Computer and Information Science
    Identifiers
    urn:nbn:se:uu:diva-12745 (URN)
    Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2018-01-12Bibliographically approved
    8. Increasing the dynamic range of in situ PLA
    Open this publication in new window or tab >>Increasing the dynamic range of in situ PLA
    Show others...
    2011 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 11, p. 892-893Article in journal, Editorial material (Refereed) Published
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-159199 (URN)10.1038/nmeth.1743 (DOI)000296891800004 ()
    Available from: 2011-09-25 Created: 2011-09-25 Last updated: 2017-12-08Bibliographically approved
    9. High-throughput cellular-resolution in vivo vertebrate screening
    Open this publication in new window or tab >>High-throughput cellular-resolution in vivo vertebrate screening
    Show others...
    2011 (English)In: Proc. 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 2011Conference paper, Published paper (Refereed)
    National Category
    Medical Image Processing
    Identifiers
    urn:nbn:se:uu:diva-159201 (URN)
    Available from: 2011-09-25 Created: 2011-09-25 Last updated: 2011-11-04
  • 2.
    Allalou, Amin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    van de Rijke, Frans
    Jahangir Tafrechi, Roos
    Raap, Anton
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    Image based measurements of single cell mtDNA mutation load MTD 20072007In: Medicinteknikdagarna 2007, 2007Conference paper (Other (popular science, discussion, etc.))
    Abstract [en]

    Cell cultures as well as cells in tissue always display a certain degree of variability,and measurements based on cell averages will miss important information contained in a heterogeneous population. These differences among cells in a population may be essential to quantify when looking at, e.g., protein expression and mutations in tumor cells which often show high degree of heterogeneity.

    Single nucleotide mutations in the mithochondrial DNA (mtDNA) can accumulate and later be present in large proportions of the mithocondria causing devastating diseases. To study mtDNA accumulation and segregation one needs to measure the amount of mtDNA mutations in each cell in multiple serial cell culture passages. The different degrees of mutation in a cell culture can be quantified by making measurements on individual cells as an alternative to looking at an average of a population. Fluorescence microscopy in combination with automated digital image analysis provides an efficient approach to this type of single cell analysis.

    Image analysis software for these types of applications are often complicated and not easy to use for persons lacking extensive knowledge in image analysis, e.g., laboratory personnel. This paper presents a user friendly implementation of an automated method for image based measurements of mtDNA mutations in individual cells detected with padlock probes and rolling-circle amplification (RCA). The mitochondria are present in the cell’s cytoplasm, and here each cytoplasm has to be delineated without the presence of a cytoplasmic stain. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%. The final image based measurements of mitochondrial mutation load are also compared to, and show high agreement with, measurements made using biochemical techniques.

  • 3.
    Allalou, Amin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    van de Rijke, Frans M.
    Jahangir Tafrechi, Roos
    Raap, Anton K.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    Image Based Measurements of Single Cell mtDNA Mutation Load2007In: Image Analysis, Proceedings / [ed] Ersboll BK, Pedersen KS, 2007, p. 631-640Conference paper (Refereed)
    Abstract [en]

    Cell cultures as well as cells in tissue always display a certain degree of variability, and measurements based on cell averages will miss important information contained in a heterogeneous population. This paper presents automated methods for image based measurements of mitochondiral DNA (mtDNA) mutations in individual cells. The mitochondria are present in the cell’s cytoplasm, and each cytoplasm has to be delineated. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%. The final image based measurements of mitochondrial mutation load are also compared to, and show high agreement with, measurements made using biochemical techniques.

  • 4.
    Allalou, Amin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Wu, Yuelong
    Ghannad-Rezaie, Mostafa
    Eimon, Peter M.
    Yanik, Mehmet Fatih
    Automated deep-phenotyping of the vertebrate brain2017In: eLIFE, E-ISSN 2050-084X, Vol. 6, article id e23379Article in journal (Refereed)
  • 5.
    Allalou, Amin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    BlobFinder, a tool for fluorescence microscopy image cytometry2009In: Computer Methods and Programs in Biomedicine, ISSN 0169-2607, E-ISSN 1872-7565, Vol. 94, no 1, p. 58-65Article in journal (Refereed)
    Abstract [en]

    Images can be acquired at high rates with modern fluorescence microscopy hardware, giving rise to a demand for high-speed analysis of image data. Digital image cytometry, i.e., automated measurements and extraction of quantitative data from images of cells, provides valuable information for many types of biomedical analysis. There exists a number of different image analysis software packages that can be programmed to perform a wide array of useful measurements. However, the multi-application capability often compromises the simplicity of the tool. Also, the gain in speed of analysis is often compromised by time spent learning complicated software. We provide a free software called BlobFinder that is intended for a limited type of application, making it easy to use, easy to learn and optimized for its particular task. BlobFinder can perform batch processing of image data and quantify as well as localize cells and point like source signals in fluorescence microscopy images, e.g., from FISH, in situ PLA and padlock probing, in a fast and easy way.

  • 6.
    Allalou, Amin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    Signal Detection in 3D by Stable Wave Signal Verification2009In: Proceedings of SSBA 2009, 2009Conference paper (Other academic)
    Abstract [en]

    Detection and localization of point-source signals is an important task in many image analysis applications. These types of signals can commonly be seen in fluorescent microscopy when studying functions of biomolecules. Visual detection and localization of point-source signals in 3D is limited and time consuming, making automated methods an important task. The 3D Stable Wave Detector (3DSWD) is a new method that combines signal enhancement with a verifier/separator. The verifier/separator examines the intensity gradient around a signal, making the detection less sensitive to noise and better at separating spatially close signals. Conventional methods such as; TopHat, Difference of Gaussian, and Multiscale Product consist only of signal enhancement. In this paper we compare the 3DSWD to these conventional methods with and without the addition of a verifier/separator. We can see that the 3DSWD has the highest robustness to noise among all the methods and that the other methods are improved when a verifier/separator is added.

  • 7.
    Allalou, Amin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    van de Rijke, Frans
    Jahangir Tafrechi, Roos
    Raap, Anton
    Segmentation of Cytoplasms of Cultured Cells2007In: In Proceedings SSBA 2007, Symposium on image analysis, Linköping, 2007Conference paper (Other academic)
    Abstract [en]

    Cell cultures as well as cells in tissue always display a certain degree of variability, and measurements based on cell averages will miss important information contained in a heterogeneous population. This paper presents automated methods for segmentation of cells and cytoplasms. The segmentation results are applied to image based measurements of mitochondiral DNA (mtDNA) mutations in individual cells. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%, compared to an inter observer variability of 79% at manual delineation.

  • 8.
    Bombrun, Maxime
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Ranefall, Petter
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Lindblad, Joakim
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Allalou, Amin
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Partel, Gabriele
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Solorzano, Leslie
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Qian, Xiaoyan
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Tomtebodavagen 23, S-17165 Solna, Sweden.
    Nilsson, Mats
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Tomtebodavagen 23, S-17165 Solna, Sweden.
    Wählby, Carolina
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Decoding gene expression in 2D and 3D2017In: Image Analysis: Part II, Springer, 2017, p. 257-268Conference paper (Refereed)
    Abstract [en]

    Image-based sequencing of RNA molecules directly in tissue samples provides a unique way of relating spatially varying gene expression to tissue morphology. Despite the fact that tissue samples are typically cut in micrometer thin sections, modern molecular detection methods result in signals so densely packed that optical “slicing” by imaging at multiple focal planes becomes necessary to image all signals. Chromatic aberration, signal crosstalk and low signal to noise ratio further complicates the analysis of multiple sequences in parallel. Here a previous 2D analysis approach for image-based gene decoding was used to show how signal count as well as signal precision is increased when analyzing the data in 3D instead. We corrected the extracted signal measurements for signal crosstalk, and improved the results of both 2D and 3D analysis. We applied our methodologies on a tissue sample imaged in six fluorescent channels during five cycles and seven focal planes, resulting in 210 images. Our methods are able to detect more than 5000 signals representing 140 different expressed genes analyzed and decoded in parallel.

  • 9.
    Chang, Tsung-Yao
    et al.
    Massachusetts Institute of Technology, USA.
    Pardo-Martin, Carlos
    Massachusetts Institute of Technology, USA.
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Yanik, Mehmet Fatih
    Massachusetts Institute of Technology, USA.
    Fully automated cellular-resolution vertebrate screening platform with parallel animal processing2012In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 12, no 4, p. 711-716Article in journal (Refereed)
    Abstract [en]

    The zebrafish larva is an optically-transparent vertebrate model with complex organs that is widelyused to study genetics, developmental biology, and to model various human diseases. In this article, wepresent a set of novel technologies that significantly increase the throughput and capabilities of ourpreviously described vertebrate automated screening technology (VAST). We developed a robustmulti-thread system that can simultaneously process multiple animals. System throughput is limitedonly by the image acquisition speed rather than by the fluidic or mechanical processes. We developedimage recognition algorithms that fully automate manipulation of animals, including orienting andpositioning regions of interest within the microscope’s field of view. We also identified the optimalcapillary materials for high-resolution, distortion-free, low-background imaging of zebrafish larvae.

  • 10.
    Clausson, Carl-Magnus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Weibrecht, Irene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mahmoudi, Salah
    Farnebo, Marianne
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Increasing the dynamic range of in situ PLA2011In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 11, p. 892-893Article in journal (Refereed)
  • 11.
    Gudmundsson, Sanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University.
    Wilbe, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Gorniok, Beata Filipek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology.
    Molin, Anna-Maja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Ekvall, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Johansson, Josefin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology.
    Gylje, Hans
    Department of Paediatrics, Central Hospital, Västerås, 721 89, Sweden..
    Kalscheuer, Vera M.
    Research Group Development and Disease, Max Planck Institute for Molecular Genetics, Berlin, 141 95, Germany..
    Ledin, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology.
    Annerén, Göran
    Bondeson, Marie-Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    TAF1, associated with intellectual disability in humans, is essential for life and regulates neurodevelopmental processes in zebrafishManuscript (preprint) (Other academic)
    Abstract [en]

    The TATA-box binding protein associated factor 1 (TAF1) protein is a key unit of the transcription factor II D complex that serves a vital function during transcription initiation. Variants of TAF1 have been associated with neurodevelopmental disorders, but TAF1’s molecular function remains elusive. In this study, we present a five-generation family affected with X-linked intellectual disability, co-segregating with a TAF1 c.3568C>T, p.(Arg1190Cys) variant. All affected males presented with intellectual disability and dysmorphic features, while carrier females were asymptomatic and had completely skewed X-chromosome inactivation. We investigated the role of TAF1 and its association to neurodevelopment during early embryogenesis by creating the first complete knockout model of the TAF1 orthologue in zebrafish. A crucial role of human TAF1 during early embryogenesis can be inferred from the model, demonstrating that an intact taf1 is essential for life from early embryonic development. Transcriptome analysis of taf1 zebrafish knockout revealed enrichment of genes in pathways associated with neurodevelopmental processes. In conclusion, we suggest that TAF1 is essential for life and that functional TAF1 is vital for early neurogenesis.

  • 12.
    Ljungblad, Jonas
    et al.
    Hok Instrument AB, S-72131 Vasteras, Sweden..
    Hök, Bertil
    Hok Instrument AB, S-72131 Vasteras, Sweden..
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Pettersson, Håkan
    Autoliv Dev AB, Vargarda, Sweden..
    Passive in-vehicle driver breath alcohol detection using advanced sensor signal acquisition and fusion2017In: Traffic Injury Prevention, ISSN 1538-9588, E-ISSN 1538-957X, Vol. 18, p. S31-S36Article in journal (Refereed)
    Abstract [en]

    Objective: The research objective of the present investigation is to demonstrate the present status of passive in-vehicle driver breath alcohol detection and highlight the necessary conditions for large-scale implementation of such a system. Completely passive detection has remained a challenge mainly because of the requirements on signal resolution combined with the constraints of vehicle integration. The work is part of the Driver Alcohol Detection System for Safety (DADSS) program aiming at massive deployment of alcohol sensing systems that could potentially save thousands of American lives annually.

    Method: The work reported here builds on earlier investigations, in which it has been shown that detection of alcohol vapor in the proximity of a human subject may be traced to that subject by means of simultaneous recording of carbon dioxide (CO2) at the same location. Sensors based on infrared spectroscopy were developed to detect and quantify low concentrations of alcohol and CO2. In the present investigation, alcohol and CO2 were recorded at various locations in a vehicle cabin while human subjects were performing normal in-step procedures and driving preparations. A video camera directed to the driver position was recording images of the driver's upper body parts, including the face, and the images were analyzed with respect to features of significance to the breathing behavior and breath detection, such as mouth opening and head direction.

    Results: Improvement of the sensor system with respect to signal resolution including algorithm and software development, and fusion of the sensor and camera signals was successfully implemented and tested before starting the human study. In addition, experimental tests and simulations were performed with the purpose of connecting human subject data with repeatable experimental conditions. The results include occurrence statistics of detected breaths by signal peaks of CO2 and alcohol. From the statistical data, the accuracy of breath alcohol estimation and timing related to initial driver routines (door opening, taking a seat, door closure, buckling up, etc.) can be estimated.The investigation confirmed the feasibility of passive driver breath alcohol detection using our present system. Trade-offs between timing and sensor signal resolution requirements will become critical. Further improvement of sensor resolution and system ruggedness is required before the results can be industrialized.

    Conclusions: It is concluded that a further important step toward completely passive detection of driver breath alcohol has been taken. If required, the sniffer function with alcohol detection capability can be combined with a subsequent highly accurate breath test to confirm the driver's legal status using the same sensor device. The study is relevant to crash avoidance, in particular driver monitoring systems and driver-vehicle interface design.

  • 13. Pardo-Martin, Carlos
    et al.
    Allalou, Amin
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Medina, Jaime
    Eimon, Peter M.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Yanik, Mehmet Fatih
    High-throughput hyperdimensional vertebrate phenotyping2013In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 4, p. 1467-Article in journal (Refereed)
    Abstract [en]

    Most gene mutations and biologically active molecules cause complex responses in animals that cannot be predicted by cell culture models. Yet animal studies remain too slow and their analyses are often limited to only a few readouts. Here we demonstrate high-throughput optical projection tomography with micrometre resolution and hyperdimensional screening of entire vertebrates in tens of seconds using a simple fluidic system. Hundreds of independent morphological features and complex phenotypes are automatically captured in three dimensions with unprecedented speed and detail in semitransparent zebrafish larvae. By clustering quantitative phenotypic signatures, we can detect and classify even subtle alterations in many biological processes simultaneously. We term our approach hyperdimensional in vivo phenotyping. To illustrate the power of hyperdimensional in vivo phenotyping, we have analysed the effects of several classes of teratogens on cartilage formation using 200 independent morphological measurements, and identified similarities and differences that correlate well with their known mechanisms of actions in mammals.

  • 14. Pardo-Martin, Carlos
    et al.
    Chang, Tsung-Yao
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Yanik, Mehmet Fatih
    High-throughput cellular-resolution in vivo vertebrate screening2011In: Proc. 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 2011Conference paper (Refereed)
  • 15.
    Pinidiyaarachchi, Amalka
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    Zieba, Agata
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    Pardali, Katerina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    A detailed analysis of 3D subcellular signal localization2009In: Cytometry Part A, ISSN 1552-4922, Vol. 75A, no 4, p. 319-328Article in journal (Refereed)
    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.

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