Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells. at 4?C for 30?min. single-molecule colocalization?analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely relevant experimental controls to determine complete fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complexconsisting of talin, kindlin and active Dantrolene sodium Hemiheptahydrate 1-integrinthat specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells. at 4?C for 30?min. The supernatant was removed and origami was resuspended in folding buffer (12.5?mM MgCl2, 10?mM Tris, 1?mM EDTA at pH 8.0). Centrifugation and supernatant removal were repeated three times. Origami was stored at ?20?C. Cell experiments with DNA origami Cells were seeded, fixed, and labeled as described above. To perform qPAINT experiments, labeling answer was removed and cells were washed 3 times with 1 PBS. Next, 200?l BSA-Biotin solution (1?mg/ml BSA-Biotin in buffer A+ (10?mM Tris-HCl, 100?mM NaCl, and 0.05% Tween-20, pH 8.0)) was added and incubated for 10?min. The dish was then cautiously washed with buffer A+, 200?l streptavidin solution (0.5?mg/ml in buffer A+) was added, Dantrolene sodium Hemiheptahydrate and incubated for another 10?min. Afterwards, the dish was washed with buffer A+ and subsequently with buffer C Dantrolene sodium Hemiheptahydrate (1 PBS?+?500?mM NaCl). Then, 200?l of biotin-labeled DNA origami answer was added (200?pM in buffer C) and incubated for 60?min. Finally, the dish was cautiously washed with buffer C and imaging buffer was added. Antibody conjugation to DNA-PAINT docking strands The integrin 1 9EG7 antibody was conjugated to DNA-PAINT docking strands using a bifunctional NHS ester crosslinker harboring an additional trans-cyclooctene moiety (TCO; TCO-NHS ester ((being the Dantrolene sodium Hemiheptahydrate density), as explained in equation (1): thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. CCL4 Publishers notice Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Lisa S. Fischer, Christoph Klingner, Thomas Schlichthaerle. Contributor Information Reinhard F?ssler, Email: ed.gpm.mehcoib@relsseaf. Ralf Jungmann, Email: ed.gpm.mehcoib@nnamgnuj. Carsten Grashoff, Email: ed.uww@ffohsarg. Supplementary information The online version contains supplementary material available at 10.1038/s41467-021-21142-2..