Disease diagnosis often relies on histopathological analysis of biopsy tissue or cells and requires high specificity and sensitivity. This poses a major challenge to current methods and necessitates the development of improved techniques that allow detailed studies of biomolecules in situ. Such studies include the detection of biomolecule abundance, their sub-cellular localisation and secondary modifications, as well as their interaction with other molecules. The main aim of the EU-funded project ‘Enhanced Ligase based histochemical techniques’ (Enlight) was to develop highly sensitive and specific analytical procedures to study individual nucleic acid and protein molecules and their functional status. The project was based on the 'proximity ligation assay' (PLA) technology for protein analysis and on 'padlock probing' for DNA analysis, two recently invented technologies that allow single bio-molecule detection in single cells and tissue in situ. Using two antibodies coupled to DNA oligonucleotides, the PLA method takes advantage of the proximity of the bound antibodies on the same protein molecule. The DNA oligonucleotides are subsequently joined together and another fluorescent DNA probe is used for detection under the microscope. The Enlight consortium refined this method to analyse individual proteins, their interactions, modifications and localisation. Additionally, by developing reagents for the detection of cancer biomarkers, project partners succeeded in gaining new biological knowledge on the role of several proteins in carcinogenesis and mitochondrial diseases. Padlock probes are DNA molecules consisting of 20-nucleotide–long segments complementary to the target connected by a 40-nucleotide–long linker sequence. They offer great sensitivity and can distinguish DNA molecules of high sequence similarity. The project partners optimised this technology to distinguish single-nucleotide differences between different mitochondrial genomes, and their localisation in situ. Quantification of the intensity of the signals generated by the above methods was performed using automated image analysis procedures. The developed software tools allowed processing of many samples and facilitated unbiased classification of molecules and their localisation in tissues or cells. Project deliverables constitute unique molecular tools for studying individual proteins or nucleic acids and their functional status in single cells and clinical specimens. Commercial exploitation of these techniques is expected to improve disease diagnosis and hopefully disease prevention.