Significance: The nanoscale organization of ligands and receptors is critical for cellular communication yet inherently challenging to investigate. We have here devised a DNA origami-based biointerface which allows the experimenter to adjust protein distances with nanometer precision as a means to enhance or disturb signaling while being responsive to large-scale reorganization processes during cell activation. Applying this biointerface to study the spatial requirements of T cell activation, we find that the smallest signaling-competent receptor unit consists of two stably ligated T cell receptors (TCRs) within a distance of 20 nanometers. Spatial organization of the physiological ligand pMHC, however, is not a relevant parameter of antigen-mediated T cell activation, as single, well-isolated pMHC molecules efficiently stimulate T cells.

Fluorescence microscopy is an invaluable tool in biology, yet its performance is compromised when the wavefront of light is distorted due to optical imperfections or the refractile nature of the sample. Such optical aberrations can dramatically lower the information content of images by degrading the image contrast, resolution, and signal. Adaptive optics (AO) methods can sense and subsequently cancel the aberrated wavefront, but they are too complex, inefficient, slow, or expensive for routine adoption by most labs. Here, we introduce a rapid, sensitive, and robust wavefront sensing scheme based on phase diversity, a method successfully deployed in astronomy but underused in microscopy. Our method enables accurate wavefront sensing to less than λ/35 root mean square (RMS) error with few measurements, and AO with no additional hardware besides a corrective element. After validating the method with simulations, we demonstrate the calibration of a deformable mirror >hundredfold faster than comparable methods (corresponding to wavefront sensing on the ∼100ms scale), and sensing and subsequent correction of severe aberrations (RMS wavefront distortion exceeding λ/2), restoring diffraction-limited imaging on extended biological samples.

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

While single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential has not yet been fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the qualitative analysis of two-dimensional SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. The method is parameter-free and does not require any additional measurements nor grouping of localizations. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers.

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to forma functional signal transducing complex. Here, weapplied singlemolecule imaging capable of detecting long-lived and mobile associations tomeasure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 coexpression, a small fraction was present as constitutive homodimers exhibiting a similar to 40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced themobility of homodimers fourfold compared to constitutive homodimers. Expression ofErbB2 elevated the homodimeric fraction ofErbB3 even in unstimulated cells and induced a similar to 2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization armofErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.

In the body, a T cell is confronted with millions of antigen-presenting cells (APCs) in the search for potentially harmful antigen. To elicit an appropriate immune response, this search has to be performed as fast and as precise as possible. These two requirements, however, are at odds with each other: fast searches lack accuracy, whereas high fidelity decisions are typically time-consuming. Here, we use the archetypical search for the needle in the haystack as an analogy for the T cell's search problem. We provide a statistical framework to quantitatively estimate the constraints of search strategies for rare instances. Particularly, we propose a solution for balancing the demand for high speed with low error rates. It takes advantage of a two-phase search process, which combines a first rapid scan with a second high-fidelity check. Finally, we provide arguments that support a two-phase search model for identification of antigen-positive APCs by T cells.