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.

Fluorescently labeled recombinant RNAP system to probe archaeal transcription initiation

“…These parameters can be used to determine the molecular state of an ensemble of molecules or for a single molecule. Single-molecule measurements are particularly well suited for the interrogation of biomolecules as they provide insights into the heterogeneity of a population and inform about the dynamics of a system often measurable in realtime without the need for synchronization of the dynamic process (for recent reviews see [60][61][62][63]). …”

“…where n is the refractive index of the medium, U 0 is the quantum yield of the donor fluorophore in the absence of the acceptor fluorophore, j 2 is the orientation factor (for a freely rotating fluorophore a value of 2/3 is assumed) and J is the integral of the spectral overlap between donor emission and acceptor absorption spectra [60,66,68].…”

Techniques for the analysis of protein-protein interactions in vivo

“…Using multiple prey proteins with different fluorophores allows for the exploration and permutation of higher order protein complexes. Additionally, combining total internal reflection microscopy with photobleaching or lifetime imaging allows for stoichiometric analysis (Gust et al, 2014). These methods have not yet been applied in the plant field but will surely provide a powerful platform in the near future.…”

Sensing Inside the Living Cells

“…13.21 . Sensing on a single molecular level is commonly limited to very high dilutions of interacting partners, down to picomolar or low nanomolar concentration range (Gust et al 2014 ). This limits the target detection to high-affi nity binding, as it was explained in Chap.…”

“…The choice of fl uorescent reporters and the mechanisms of their response in sensing have to be the most carefully selected (Gust et al 2014 ). The perylene 3,4,9,10-tetracarboxyl bisimide was suggested as a very prospective dye that can be excited by 488 nm line of argon ion laser and exhibits almost 100 % quantum yield.…”