||Computerized image analysis is applied in biomedicine to control industrial processes or extract information required for medical diagnostics. In this seminar I will present another application of image analysis in method development of fundamental biochemistry tools.
Padlock probes and proximity ligation assays are just a few examples of several important tools developed at the Faculty of Medicine in Uppsala in recent decades. A target signal, which could be a DNA-sequence (padlock probes) or a protein of interest (proximity ligation assays) could be labeled by one, two or more fluorescent labels. Ideally, each individual interaction of biomolecules is amplified into a measurable point-like signal by rolling-circle amplification.
Once the specimen is prepared, wide-field fluorescence microscopy and spectral image analysis are employed to evaluate the quality of multi-coloured signals. The image analysis pipeline consists of three main parts - correction of optical aberrations, signal detection and colour-based signal classification. Finally, evaluation of image analysis methods will be presented, as well as several applications in biotechnology: verification of signal detection and classification using dilution series, estimation of "biochemical noise", comparison of synthetic and clonal oligonucleotides and quantification of DNA-protein interactions.