Knowledge of how proteins organize into functional complexes is essential to understand their biological function. Optical super-resolution techniques provide the spatial resolution necessary to visualize and to investigate individual protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) builds on the detection of single fluorophore labels, which next to the generation of high-resolution images provides access to quantitative molecular information. We developed various tools for quantitative SMLM (qSMLM), an imaging method that both super-resolves individual protein clusters and reports on molecular numbers by analyzing the kinetics of single emitter blinking. This method is compatible with both fluorescent proteins and organic fluorophores. With qSMLM, we quantify protein copy numbers in single clusters, and we study how changes in the stoichiometry of protein complexes translates into function.
Correlating DNA-PAINT (point accumulation for imaging in nanoscale topography) and single-molecule FRET (Förster resonance energy transfer) enables the multiplexed detection with sub-diffraction optical resolution. We designed pairs of short oligonucleotides, labeled with donor and acceptor fluorophores with various distances generating different FRET efficiencies. The strands can transiently bind to a target docking strand, simultaneous binding of both strands results in FRET signals which yield a super-resolved image via DNA-PAINT imaging. We demonstrate FRET-PAINT by designing and imaging DNA origami, which is a useful tool to establish super-resolution methods. The DNA origami structures were equipped with three target binding sites spaced by 55 nm, a sub-diffraction limited distance, however ensuring that no FRET between the target sites occurs. We resolved the individual binding sites in the donor and acceptor channels, and in addition extracted the FRET efficiency for each site in single and mixed populations. The combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging in conjunction with distance-sensitive readout in the 1 to 10 nm range.
Knowledge of assembly, subunit architecture and dynamics of membrane proteins in a cellular context is essential to infer their biological function. Optical super-resolution techniques provide the necessary spatial resolution to study these properties of membrane protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) is particularly well suited, as next to high-resolution images, it provides quantitative information on the detection of single emitters. A challenge for current super-resolution methods is to resolve individual protein subunits within a densely packed protein cluster. For this purpose, we developed quantitative SMLM (qSMLM), which reports on molecular numbers by analyzing the kinetics of single emitter blinking. Next to theoretical models for various photophysical schemes, we demonstrate this method for a selection of fluorescent proteins and synthetic dyes and a selection of membrane proteins. We next applied this tool to toll-like receptor 4 (TLR4), and found a ligand-specific formation of monomeric or dimeric receptors. Next to fluorescent proteins, DNA-PAINT offers a novel and flexible approach for quantitative super-resolution microscopy. We demonstrate DNA-PAINT imaging of structurally defined DNA origami structures and robust quantification of target sites, as well as of membrane receptors. Molecular quantification, together with experiments following single receptor mobilities in live cells, will enlighten molecular mechanisms of receptor activation.
We report on quantitative single-molecule localization microscopy, a method that next to super-resolved images of cellular structures provides information on protein copy numbers in protein clusters. This approach is based on the analysis of blinking cycles of single fluorophores, and on a model-free description of the distribution of the number of blinking events. We describe the experimental and analytical procedures, present cellular data of plasma membrane proteins and discuss the applicability of this method.