title: A starting point for fluorescence-based single-molecule measurements in biomolecular research.
abstract: Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.
authors.affiliation: Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, Braunschweig 38106, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Gust
authors.affiliation: Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, Braunschweig 38106, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Zander
authors.affiliation: Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, Braunschweig 38106, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gietl
authors.affiliation: Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, Braunschweig 38106, Germany.
authors.forename: Phil
authors.initials: P
authors.lastname: Holzmeister
authors.affiliation: Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, Braunschweig 38106, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Schulz
authors.affiliation: Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, Braunschweig 38106, Germany.
authors.forename: Birka
authors.initials: B
authors.lastname: Lalkens
authors.affiliation: Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, Braunschweig 38106, Germany.
authors.forename: Philip
authors.initials: P
authors.lastname: Tinnefeld
authors.affiliation: Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, Braunschweig 38106, Germany. d.grohmann@tu-bs.de.
authors.forename: Dina
authors.initials: D
authors.lastname: Grohmann
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D012106:Research; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005456:Fluorescent Dyes