title: Let There Be Light!
abstract: 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 Förster resonance energy transfer (FRET) andin situproximity ligation assay (in situPLA) 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.
authors.affiliation: Department of Pharmaceutical Biosciences, Pharmaceutical Cell Biology, Biomedical Center, Box 594, Uppsala University, SE-751 08 Uppsala, Sweden. doroteya.raykova@igp.uu.se.
authors.forename: Doroteya
authors.initials: D
authors.lastname: Raykova
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany. bjoern.koos@mpi-dortmund.mpg.de.
authors.forename: Björn
authors.initials: B
authors.lastname: Koos
authors.affiliation: Department of Immunology, Genetics and Pathology, Dag Hamarskjölds väg 14B, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden. anna.asplund@igp.uu.se.
authors.forename: Anna
authors.initials: A
authors.lastname: Asplund
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany. marton.gelleri@mpi-dortmund.mpg.de.|Faculty of Chemistry and Chemical Biology, Technical University of Dortmund, Otto-Hahn-Straße 6, 44221 Dortmund, Germany. marton.gelleri@mpi-dortmund.mpg.de.
authors.forename: Márton
authors.initials: M
authors.lastname: Gelléri
authors.affiliation: Department of Chemistry-BMC, Box 576, Uppsala University, SE-751 23 Uppsala, Sweden. ylva.ivarsson@kemi.uu.se.
authors.forename: Ylva
authors.initials: Y
authors.lastname: Ivarsson
authors.affiliation: Department of Chemistry-BMC, Box 576, Uppsala University, SE-751 23 Uppsala, Sweden. helena.danielson@kemi.uu.se.
authors.forename: U Helena
authors.initials: UH
authors.lastname: Danielson
authors.affiliation: Department of Pharmaceutical Biosciences, Pharmaceutical Cell Biology, Biomedical Center, Box 594, Uppsala University, SE-751 08 Uppsala, Sweden. ola.soderberg@igp.uu.se.
authors.forename: Ola
authors.initials: O
authors.lastname: Söderberg
keywords: FRET; high resolution microscopy; in situ PLA; post-translational modifications; protein–protein interactions; proxHCR