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.

We report the development of multiplexed cellular super-resolution imaging using DNA-barcoded binders.


Microscope setup
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 and 1000 nM DNA-PAINT docking strands in folding buffer (1× TE buffer with 12.5 mM MgCl 2 ). The solution was annealed using a thermal ramp cooling from 90 °C to 4 °C over the course of 3 h.
Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2017

DNA origami sample preparation and imaging
For sample preparation, a piece of coverslip (No. 1.5,18 × 18 mm ,0.17 mm thick) and a glass slide (3 × 1 inch 2 , 1 mm thick) were sandwiched together by two strips of double-sided tape to form a flow chamber with inner volume of ≈20 µl. First, 20 µl of biotin-labeled bovine albumin (Sigma A8549, 1 mg/ml, dissolved in buffer A) was flown into the chamber and incubated for 2 min. The chamber was then washed using 40 µl of buffer A. 20 µl of streptavidin (Thermo, S888, 0.5 mg/ml, dissolved in buffer A) was then flown through the chamber and was allowed to bind for 2 min. After washing with 40 µl of buffer A and subsequently with 40 µl of buffer B, 20 µl of a mix of all 51 DNA origami multiplexing structures in buffer B (dilution of 1 in 25) were finally flown into the chamber and incubated for 2 min. The chamber was washed using 40 µl of buffer B. The final imaging buffer solution contained 3 nM ATTO655-labeled (P1) imager strands and 6 nM ATTO655-labeled (Pi,i ϵ [2,52]) imager strands in buffer B. The chamber was sealed with epoxy before subsequent imaging. The EMCCD readout bandwidth was set to 3 MHz (no EM Gain) at 16 Bit. Imaging was performed using oblique illumination with an excitation intensity of ~300 W/cm 2 at 642 nm. Images were acquired for 10000 frames (200 ms integration time, total imaging time ~33 min).

EGFR Transfection
At confluence, before plating, cells were washed, trypsinized and suspended in culture media. Cells were counted. In a typical experiment, ~50000 cells/well were plated in 8-well Nunc™ Lab-Tek™ Chamber Slides. 24 h post plating, when the cells achieved ~70% confluency, 2 μg or 4 μg of EGFR plasmid DNA along with P3000 were mixed together in 5 μl of Opti-MEM for each well in the chamber. At the same time, Lipofectamine 3000 transfection agent was mixed separately in 10 μl of Opti-MEM. The Lipofectamine and the DNA reagents were mixed in a 1:1 ratio and incubated at room temperature for 5 minutes to form complexes. This was added dropwise to cells and the cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. After 24 h, the media was replaced with Dulbecco's Modified Eagle Medium (DMEM), supplemented with fetal bovine serum (FBS; 10%), penicillin and streptomycin (1%), and L-glutamine (1%). Typically 48 h following transfection cells were used in the indicated assays.

CellLight Mitochondria-GFP, BacMam 2.0 Transfection
At confluence, before plating, HeLa cells were washed, trypsinized and suspended in culture medium. Cells were counted and ~50000 cells were plated in a Labtek Chamber. After 24 h, 10 μl of CellLight Mitochondria-GFP, BacMam 2.0 Transfection reagent was added to each well in the chamber and cells were incubated at 37 °C in a humidified atmosphere containing 5% CO 2 . The transfection efficiency was checked 24 h after transfection and the cells were used typically 48 h after transfection.
1. 24 h before incubation, ~25,000 cells/well was plated in a Lab-Tek chamber.
2. Culture media was removed and proceed to fixation.
1. 24 h before incubation, ~25,000 cells/well was plated in a Lab-Tek chamber.
2. Culture media was removed and proceed to fixation.
6. Staining for overnight at 4 °C with primary antibody (10 μg/ml) against tubulin diluted in 3% bovine serum albumin and 0.1% v/v Triton X-100 in PBS to a concentration of 10 µg/mL. 7. Washing with PBS (3X) with 5 min incubation each time.
10. Proceed to DNA-PAINT imaging.  Each origami contains a unique 6-bit barcode addressable with imager sequence P1 (left side), and single-stranded extensions that will act as docking sites for the imager to be tested (P2 -P52). Together, these extensions form a mirrored "F" shape (right side).