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

Direct link
Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Show others and affiliations
2007 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 55, no 11, 1159-1166 p.Article 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.

Place, publisher, year, edition, pages
2007. Vol. 55, no 11, 1159-1166 p.
Keyword [en]
A3243G mtDNA, Aging, Heteroplasmy, Mitochondrial diseases, Mutation load, Padlock probing, PCR-RFLP, Segregation
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-12658DOI: 10.1369/jhc.7A7282.2007ISI: 000250320100009PubMedID: 17679731OAI: oai:DiVA.org:uu-12658DiVA: diva2:40427
Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2011-11-04Bibliographically 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 856
Image analysis, cytmetry, model organism, zebrafish, screening
National Category
Medical Image Processing
Research subject
Computerized Image Processing
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)
Available from: 2011-10-21 Created: 2011-09-25 Last updated: 2014-07-21Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Larsson, ChatarinaWählby, Carolina
By organisation
Centre for Image AnalysisComputerized Image AnalysisDepartment of Genetics and Pathology
In the same journal
Journal of Histochemistry and Cytochemistry
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 232 hits
ReferencesLink to record
Permanent link

Direct link