Let There Be Light!

The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Forster resonance energy transfer (FRET) and in situ proximity ligation assay (in situ PLA) to monitor protein interactions. Microscopy has become a commonly used method for the investigation of molecular events within the cell, for the identification of key players in signaling networks, and the activation of these pathways. Multiple approaches are available for functional analyses in single cells. They provide information not only on the localization of proteins at a given time point, but also on their expression levels and activity states, allowing us to pinpoint hallmarks of different cellular identities within tissues in health and disease. Clever solutions to increase the sensitivity of molecular tools, the possibilities for multiplexing, as well as image resolution have recently been introduced; however, these methods have their pros and cons. Therefore, one needs to carefully consider the biological question of interest along with the nature of the sample before choosing the most suitable method or combination of methods. Herein, we review a few of the most exciting microscopy-based molecular techniques for proteomic analysis and cover the benefits as well as the disadvantages of their use

Optimization of proximity-dependent initiation of hybridization chain reaction for improved performance

Proximity based detection methods are invaluable tools in the field of molecular biology, increasing selectivity and allowing for analysis of protein interactions. ProxHCR utilizes pairs of antibodies labelled with oligonucleotides to probe for proximal binding and to initiate a hybridization chain reaction (HCR) to generate an amplified detection signal. As HCR is based upon hybridization of DNA hairpins, the performance is dependent on salt concentrations and temperature. Herein we have redesigned the proxHCR system to increase the performance and to reduce dependency on temperature and salt concentrations. The new oligonucleotides provide an increased signal when performed at physiological salt concentrations and in room temperature