Again vimentin-mCherry-peptDTWV and TOMM20-mCherry-peptDTWV were expressed and fixed in COS7 cells
Again vimentin-mCherry-peptDTWV and TOMM20-mCherry-peptDTWV were expressed and fixed in COS7 cells. binding kinetics remains unaltered upon fixation. Finally, by fusion of our probe to nanobodies against conventional expression markers, we show that NITD008 this approach provides a versatile addition to the NITD008 super-resolution toolbox. = (= 1) leads to = PLD1 1000, equally divided over four acquisitions). Solid line shows the result of a fit with a Gaussian model. (c) Representative PAINT reconstruction of peptDTWV decorated nanoparticles imaged at low PDZ-mNG concentrations (0.1 nM) so that NITD008 single binding events could be observed for qPAINT. (d) Normalized frequency distributions for the number of biotin-peptDTWV peptides docked to a single nanoparticle (= 449). The solid line shows the result of a fit with a Gaussian model to the data to determine the mean and standard deviation (1690 712). Details of the experimental setup and conditions can be found in Supporting Information (Materials and Methods) and Table S2. Scale bar: 500 nm (a), 1 m (c). Having determined em k /em ON, we next performed qPAINT on the nanoparticles to quantify the number of accessible, peptDTWV-tagged streptavidin complexes on their surface. Imaging was performed under identical illumination conditions to those of the calibration and now in the presence of 0.1 nM PDZ-mNG to detect single binding events (Figure ?Figure22c). Measurements of the average dark times per nanoparticle resulted in an average of 1690 712 sites per nanoparticle which corresponds to 422 streptavidin tetramers (Figure ?Figure22c,d, Figure S4cCe). These values therefore indicate an approximate streptavidin spacing of 25 nm on the surface of the 300 nm nanoparticles. Interestingly, these values are in close agreement with previous bulk measurements of the accessible biotin-binding sites on commercial streptavidin-coated magnetic nanoparticles of 500 nm,39 yet higher than previously reported for DNA based qPAINT experiments.9 One possible reconciliation for this observation is a differential occupation of tetrameric streptavadin by the peptDTWV or DNA sequences as DNA is generally bigger and highly charged causing steric and electrostatic hindrance. So far, our results show that the interaction of PDZ-mNG with its peptDTWV ligand is compatible with (q)PAINT to resolve the nanoscale structure of molecular assemblies and provide quantitative information about the number of labeled molecules. Next, we wanted to explore whether this interaction remains functional after genetic expression in complex cellular environments followed by chemical fixation. This direct fusion of the peptDTWV sequence would be highly advantageous over previous strategies, as it directly controls stoichiometry and does not require additional specific targeting agents (e.g., antibodies, affimers, nanodies) or labeling steps. As conventional methods to preserve the nanoscale structure of different cellular components contain either paraformaldehyde (PFA) or a combination of PFA and glutaraldehyde (GA), targets that require fixation via either of these methods were selected. For PFA, the cytoskeletal filament vimentin was chosen23 and for PFA/GA fixation the mitochondrial outer membrane transporter subunit TOMM2040 was selected. To perform peptide-PAINT with the PDZ-mNG probe, both targets were cloned as fusion constructs with a C-terminal peptDTWV sequence. Additionally, to identify the transfected cells and have a diffraction limited reference, mCherry was engineered in between the C-terminus of these proteins and the peptDTWV. After 1 day of expression with either construct in COS7 cells, the cells were fixed and permeabilized to allow accessibility of the purified PDZ-mNG probe from solution to bind to the expressed peptDTWV sequence. Subsequently, the probe was washed in at high concentrations and imaged at high laser powers so that single binding events could be detected with high localization accuracy and frequency during 3D-PAINT acquisitions. These acquisitions resulted in high-quality super-resolved reconstructions of both cellular vimentin and TOMM20 under both fixation conditions (Figure ?Figure33). Further assessment of the image quality showed that the NITD008 PDZ-mNG/peptDTWV interaction allows for single-molecule localization with high precision (15.4 8.5 nm) to achieve an estimated Fourier ring correlation (FRC) image resolution of 53 4 nm (Figure S5). Additionally, in our approach, very little aspecific interactions of the PDZ-mNG could be observed in the fixed cellular samples which is favorable for optimal.