uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Increasing the dynamic range of in situ PLA
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.
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.
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.
(Department of Oncology-Pathology, Cancercentrum Karolinska, Karolinska Institutet.)
Show others and affiliations
2011 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 11, 892-893 p.Article in journal, Editorial material (Refereed) Published
Place, publisher, year, edition, pages
2011. Vol. 8, no 11, 892-893 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-159199DOI: 10.1038/nmeth.1743ISI: 000296891800004OAI: oai:DiVA.org:uu-159199DiVA: diva2:443417
Available from: 2011-09-25 Created: 2011-09-25 Last updated: 2017-12-08Bibliographically approved
In thesis
1. 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
2. Making Visible the Proximity Between Proteins
Open this publication in new window or tab >>Making Visible the Proximity Between Proteins
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms.

In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment.  

In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules.

The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection.

The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 973
Keyword
Proximity ligation assay, In situ PLA, rolling circle amplification, protein interaction, protein-protein interaction, in situ, single cell, single molecule, protein complex, antibody, cancer, tissue section, microscopy, image analysis, system biology, multiplex, dynamic range, methods development, systems biology
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology Biochemistry and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-217772 (URN)978-91-554-8878-9 (ISBN)
Public defence
2014-03-28, B8, Biomedicinskt centrum, Husargatan 3, SE-75123 Uppsala, 09:00 (English)
Opponent
Supervisors
Funder
Knut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme, 259796EU, FP7, Seventh Framework Programme, 278568
Available from: 2014-03-06 Created: 2014-02-04 Last updated: 2014-04-29Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Authority records BETA

Clausson, Carl-MagnusAllalou, AminLandegren, UlfWählby, CarolinaSöderberg, Ola

Search in DiVA

By author/editor
Clausson, Carl-MagnusAllalou, AminLandegren, UlfWählby, CarolinaSöderberg, Ola
By organisation
Molecular toolsScience for Life Laboratory, SciLifeLabCentre for Image AnalysisComputerized Image Analysis and Human-Computer Interaction
In the same journal
Nature Methods
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 901 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf