CAR and TCR form individual signaling synapses and do not cross-activate, however, can co-operate in T cell activation.

In engineered T cells the CAR is co-expressed along with the physiological TCR/CD3 complex, both utilizing the same downstream signaling machinery for T cell activation. It is unresolved whether CAR-mediated T cell activation depends on the presence of the TCR and whether CAR and TCR mutually cross-activate upon engaging their respective antigen. Here we demonstrate that the CD3ζ CAR level was independent of the TCR associated CD3ζ and could not replace CD3ζ to rescue the TCR complex in CD3ζ KO T cells. Upon activation, the CAR did not induce phosphorylation of TCR associated CD3ζ and, vice versa, TCR activation did not induce CAR CD3ζ phosphorylation. Consequently, CAR and TCR did not cross-signal to trigger T cell effector functions. On the membrane level, TCR and CAR formed separate synapses upon antigen engagement as revealed by total internal reflection fluorescence (TIRF) and fast AiryScan microscopy. Upon engaging their respective antigen, however, CAR and TCR could co-operate in triggering effector functions through combinatorial signaling allowing logic "AND" gating in target recognition. Data also imply that tonic TCR signaling can support CAR-mediated T cell activation emphasizing the potential relevance of the endogenous TCR for maintaining T cell capacities in the long-term.

Three-Dimensional Single Molecule Localization Microscopy Reveals the Topography of the Immunological Synapse at Isotropic Precision below 15 nm.

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. Synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. It is hence important to determine the 3D topography of the immunological synapse at high precision. Current methods provide only rather coarse images of the protein distribution within the synapse. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows three-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and nonactivating bilayers, respectively.

Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence.

The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle fluorescence (SAF), can strongly improve the axial precision, thus facilitating almost isotropic localization performance. Specific detection systems, introduced as Supercritical angle localization microscopy (SALM) or Direct optical nanoscopy with axially localized detection (DONALD), have been developed to exploit SAF in modified two-channel imaging schemes. Recently, our group has shown that off-focus microscopy, i.e., imaging at an intentional slight defocus, can perform equally well, but uses only a single detection arm. Here we compare SALM, off-focus imaging and the most commonly used 3D SMLM techniques, namely cylindrical lens and biplane imaging, regarding 3D localization in close proximity to the coverslip. We show that all methods gain from SAF, which leaves a high detection NA as the only major key requirement to unlock the SAF benefit. We find parameter settings for cylindrical lens and biplane imaging for highest z-precision. Further, we compare the methods in view of robustness to aberrations, fixed dipole emission and double-emitter events. We show that biplane imaging provides the best overall performance and support our findings by DNA-PAINT experiments on DNA-nanoruler samples. Our study sheds light on the effects of SAF for SMLM and is helpful for researchers who plan to employ localization-based 3D nanoscopy close to the coverslip.

Erratum: Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy: erratum.

[This corrects the article on p. 775 in vol. 11, PMID: 32206395.].

Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy.

Single molecule localization microscopy (SMLM) is one of the key techniques that break the classical resolution limit in optical imaging. It is based on taking multiple recordings of a sample, each showing only a sparse arrangement of spatially well separated fluorescent molecules which can be localized at nanometer precision. While localizing along the lateral directions is usually straightforward, estimating axial positions at a comparable precision is known to be much harder, which is due to the relatively large depth of focus provided by the microscope optics. Whenever a molecule is sufficiently close to the coverslip, it becomes feasible to draw additional information from near field coupling effects: super-critical angle fluorescence (SAF) appears and can be exploited to boost the axial localization precision. Here we propose defocused imaging as a SMLM strategy that is capable of leveraging the information contained in SAF. We show that, regarding axial localization precision, our approach is superior to established SAF-based approaches. At the same time it is simple and can be conducted on any research-grade microscope where controlled defocusing on the order of a few hundred nanometers is possible.