DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc05420j Click here for additional data file.

Materials Unless otherwise stated, all chemicals and solvents were purchased from commercial suppliers and used as received. PEGylated SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) cross-linker (SM(PEG) 2), trans-4-Cycloocten-1-yl 2,5-dioxo-1-pyrrolidinyl carbonate (TCO-NHS) and benzylamino tetrazine N-hydroxysuccinimidyl ester (Tz-NHS) were purchased from Sigma-Aldrich. Commercial sources of the antibodies were listed in the Supplementary Tables 3, 4, and 6. GFP nanobodies were purchased from ChromoTek. Amicon Ultra Centrifugal Filter (100 kDa MWCO) was purchased from Merck Millipore. Zeba spin desalting column (7000 MWCO) was purchased from Thermo Fisher Scientific. NAP-5 columns were purchased from GE Healthcare. Dulbecco's Phosphate-Buffered Saline (PBS, pH 7.4) without calcium and magnesium was purchased from Life Technologies. Unmodified, dye-labeled and biotinylated DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT). Streptavidin was purchased from Invitrogen (catalog number: S-888). BSA-biotin was obtained from Sigma-Aldrich (catalog number: A8549). Coverslips were purchased from VWR (coverslips 18 × 18 mm, #1.5). The glass slides were purchased from VWR (25 × 75 × 1 mm). M13mp18 scaffold was obtained from New England BioLabs (N4040s). Freeze 'N Squeeze columns were ordered from Bio-Rad (catalog number: 7326165). Fluorescence imaging was carried out on an inverted Nikon Eclipse Ti microscope (Nikon Instruments) with the Perfect Focus System, applying an objective-type TIRF configuration with an oil-immersion objective (CFI Apo TIRF 100×, NA 1.49, Oil). For excitation of ATTO655 fluorophores, a 639 nm laser (150 mW nominal, Toptica iBeam Smart) was used. The laser beam was passed through a cleanup filter (ZET 642/20x, Chroma Technology, Bellows Falls, VT) and coupled into the microscope objective using a single-band beam splitter (ZT647rdc, Chroma Technology). Fluorescence light was spectrally filtered with an emission filter (ET6651p, ET705lp, Chroma Technology). For excitation of Cy3B fluorophores, a 561 nm laser (200 mW nominal, Coherent Sapphire) was used. The laser beam was passed through a cleanup filter (ZET 561/10x, Chroma Technology, Bellows Falls, VT) and coupled into the microscope objective using a single-band beam splitter (ZT561rdc, Chroma Technology). Fluorescence light was spectrally filtered with an emission filter (ET600/50n, Chroma Technology). Single molecule fluorescence signals were imaged on an EMCCD camera (iXon Ultra 897 EMCCD, Andor Technology). Data acquisition was performed without additional magnification in the detection path and yielding a pixel size of 160 nm. DNA origami self-assembly DNA origami for crosstalk experiments were formed in a one-pot reaction with a 40 µl total volume containing 10 nM scaffold strand (M13mp18), 100 nM folding staples, 12 nM biotin-modified staples …