Quantification of colocalization and cross-talk based on spectral angles
2009 (English)In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 234, no 3, 311-324 p.Article in journal (Refereed) Published
Common methods for quantification of colocalization in fluorescence microscopy typically require cross-talk free images or images where cross-talk has been eliminated by image processing, as they are based on intensity thresholding. Quantification of colocalization includes not only calculating a global measure of the degree of colocalization within an image, but also a classification of each image pixel as showing colocalized signals or not. In this paper, we present a novel, automated method for quantification of colocalization and classification of image pixels. The method, referred to as SpecDec, is based on an algorithm for spectral decomposition of multispectral data borrowed from the field of remote sensing. Pixels are classified based on hue rather than intensity. The hue distribution is presented as a histogram created by a series of steps that compensate for the quantization noise always present in digital image data, and classification rules are thereafter based on the shape of the angle histogram. Detection of colocalized signals is thus only dependent on the hue, making it possible to classify also low-intensity objects, and decoupling image segmentation from detection of colocalization. Cross-talk will show up as shifts of the peaks of the histogram, and thus a shift of the classification rules, making the method essentially insensitive to cross-talk. The method can also be used to quantify and compensate for cross-talk, independent of the microscope hardware.
Place, publisher, year, edition, pages
Oxford, UK: Blackwell Publishing , 2009. Vol. 234, no 3, 311-324 p.
Colocalization, cross-talk, fluorescence microscopy, image analysis
Computer and Information Science
Research subject Computerized Image Analysis
IdentifiersURN: urn:nbn:se:uu:diva-111376DOI: 10.1111/j.1365-2818.2009.03170.xISI: 000266180400011PubMedID: 19493110OAI: oai:DiVA.org:uu-111376DiVA: diva2:281409
ProjectsEU-Strep project ENLIGHT (ENhanced LIGase based Histochemical Techniques)