We thank the Deutsche Forschungsgemeinschaft (DFG) for their financial support (EXC81, SFB623). We also acknowledge Stephen Hashmi (Heidelberg University) for fruitful discussions. Volker Huch is gratefully acknowledged for X-ray crystallography. Michael Schwering and Dominik Brox have continuously supported the project with their expertise in microscopy.Supporting information for this article, including details of reagents used, instruments, and analytical data, including spectroscopic characterization, is available on the WWW under http://dx.

Revolution in resolution: Abbe's resolution limit has been overcome in fluorescence microscopy by using light‐driven processes to switch the emission of fluorophores on and off. Alternatively, chemical reactions can be used, for example the coordination of Cu2+ ions to a fluorescent probe for the stochastic switching between spectroscopic states.

Obtaining quantitative information about molecular assemblies with high spatial and temporal resolution is a challenging task in fluorescence microscopy. Single-molecule techniques build on the ability to count molecules one by one. Here, a method is presented that extends recent approaches to analyze the statistics of coincidently emitted photons to enable reliable counting of molecules in the range of 1-20. This method does not require photochemistry such as blinking or bleaching. DNA origami structures are labeled with up to 36 dye molecules as a new evaluation tool to characterize this counting by a photon statistics approach. Labeled DNA origami has a well-defined labeling stoichiometry and ensures equal brightness for all dyes incorporated. Bias and precision of the estimating algorithm are determined, along with the minimal acquisition time required for robust estimation. Complexes containing up to 18 molecules can be investigated non-invasively within 150 ms. The method might become a quantifying add-on for confocal microscopes and could be especially powerful in combination with STED/RESOLFT-type microscopy.

Over the past decade, a vast variety of different fluorescent labeling systems have emerged for use in fluorescence microscopy and fluorescence-based analytical techniques. A difficulty frequently arising when quantifying fluorescently labeled samples is that the number of labels per protein is neither well defined, for example, due to multiple functional groups that can undergo covalent coupling with activated dyes, nor well known, for example, due to limited methods mostly estimating ensemble averages. Herein, we use a recently established method that evaluates the statistics of multiple photon detection events to measure the label number distribution of different fluorescent marker molecules at the single-molecule level. We tested five different far-red dyes frequently used for fluorescence labeling and found all of them suitable for our counting method. We used two dyes, ATTO647N and Alexa647, to investigate the label number distribution of fluorescently labeled proteins. In the experiments, we found that the label number distribution of antibodies and streptavidin has a significant fraction of molecules labeled with two, three, or more fluorophores. In contrast, the distribution of label numbers for nanobodies resembles the one acquired for SNAP-tag, which can have a maximum of one label per protein. This is also reflected in the ensemble degree of labeling, which is in good agreement for the latter samples, whereas stronger deviations were observed for antibodies and streptavidin. Our single-molecule studies enable full characterization of the label number distribution for various fluorescent markers. This work puts quantitative studies on the stoichiometry of fluorescently tagged oligomers and protein aggregates into perspective.

Tracking black dots placed near the inner canthi is an effective method of compensation for horizontal, vertical, and torsional in-plane head movements during pupil and iris crypt-based video-oculography.