Nonspecific binding of 10 nm Cy5-labeled DinB on nine different surfaces, measured by the number of DinB spots over an imaging area of 2,500 µm 2.

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1 Supplementary Figure 1 Nonspecific binding of 10 nm Cy5-labeled DinB on nine different surfaces, measured by the number of DinB spots over an imaging area of 2,500 µm 2. Free DinB was washed out using 50 µl imaging buffer after incubation. Numbers on the x-axis indicate different surfaces: 1) PEG; 2) PEG-siloxane-Tween-20 (ref. 12); 3) PEG-siloxane-F-127; 4) siloxane-tween-20; 5) siloxane-f-127; 6) PEG-DDS-Tween-20; 7) PEG- DDS-F-127; 8) DT20*; 9) DDS-F-127 (ref. 13). Error bars indicate s.d. DT20 was chosen for its best passivation performance as well as lowest preparation time and agent cost (see online methods).

2 Supplementary Figure 2 The background fluorescence spot counts in both Cy3 and Cy5 channels on the PEG and DT20 surfaces. The laser power was adjusted for visualization of single organic fluorophores. The appearing spots are from the impurities on the surface and their intensity is comparable to single organic fluorophores. The scale bars indicate 9 µm.

3 (a) (b) Supplementary Figure 3 The higher surface biotin density on the PEG surface and its effect on nonspecific binding. (a) Fluorescence images of biotin-bound Cy5-labeled NeutrAvidin on both surfaces. To achieve the similar surface density of NeutrAvidin, a 5 times higher concentration of Cy5-labeled NeutrAvidin was used for the DT20 surface than for the PEG surface, indicating the PEG surface has a higher surface density of biotin groups than the DT20 surface. The scale bars indicate 9 µm. (b) Passivation capacity of the PEG surface at different surface NeutrAvidin densities, measured by the average number of spots over an imaging area of 2500 µm 2 with 50 nm DinB. The 1 NeutrAvidin stock contains 0.2 mg/ml NeutrAvidin in T50 buffer (ph 8.0). Error bars indicate s.d.

4 Supplementary Figure 4 Fluorescence images of 50 nm Cy5-labeled DinB nonspecifically bound on the DDS-BSA and DT20 surfaces. Here the DDS-BSA surface refers to a DDS-coated surface that was non-specifically passivated by BSA molecules. The only difference between these two surfaces is that the DT20 surface was subjected to an additional Tween-20 passivation treatment. Other conditions, such as the BSA incubation time and concentration were the same. It suggests that the passivation effect is due to Tween-20 rather than the BSA molecules on the surface. The scale bars indicate 9 µm.

5 Supplementary Figure 5 Fluorescence images of nonspecifically bound Cy5-labeled DinB in the same area of the DT20 surface without embedded BSA or NeutrAvidin. 100 nm DinB was injected in the flow chamber and washed away after 3 min incubation. And then image (a) was recorded. The laser was kept on until all the fluorescent spots in image (a) were photobleached. Another 100 nm DinB was injected and washed away after 3 min incubation. The same area could be imaged for the second time, as shown in image (b). Images (a) and (b) are false-colored in green and red respectively and their overlay is shown in image (c). Only a few colocalized spots marked by white circles were observed. The scale bars indicate 9 µm.

6 Supplementary Figure 6 Fluorescence images of nonspecifically bound Rep-Cy5 on the DT20 and PEG surfaces at 5 nm concentration. The scale bars indicate 9 µm.

7 a b c Supplementary Figure 7 STED images of surface-bound proteins on the PEG and DT20 surfaces. (a,b) STED images of the surface-bound streptavidin-alexa 594 on both surfaces. (a) Fluorescence images of the surface (DT20) immobilized streptavidin-alexa 594 by confocal and STED microscope before the injection of 100 nm fluorescently labeled molecules. (b) Non-specific binding tests of the DT20 and PEG surfaces after washing out 100 nm streptavidin-alexa 594 by STED microscope. (c) Non-specific binding tests of the DT20 and PEG surfaces in the presence of 50 nm Alexa 594-labeled protein G B1 domain (PG-A594) by confocal and STED microscope.

8 Supplementary Figure 8 Fluorescence images of nonspecifically bound Cy3-labeled ssdna (34 nt) at 160 nm and ph 8.0. The scale bar indicates 9 µm.

9 Supplementary Figure 9 Nonspecific binding of 500 nm Abberior Star 635 labeled ssdna (18 nt) at different MgCl 2 concentrations on the DT20 surface. Error bars indicate s.d.

10 Supplementary Figure 10 Typical fluorescence intensity and FRET efficiency traces of HJ dynamics at 40 mm Mg 2+ on the PEG and DT20 surfaces. The transitions between the two FRET efficiency levels represent the transitions of HJ between its two stacked states.

11 Supplementary Figure 11 FRET histograms of single HJs at different Mg 2+ concentrations. The three peaks in the histograms correspond to, from low to high in the FRET efficiency, the Cy3 (donor) only molecules and the two stacked conformations of dual labeled HJ. The peak positions are denoted as X1, X2 and X3 while the ratio between peak 2 and 3 is denoted as Y2/Y3. The peak positions and the ratio between the low and high FRET peaks are identical between the two surfaces.

12 (a) (b) Supplementary Figure 12 The dynamics of the ribosomal protein S4-rRNA complex. (a) The schematics showing the FRET changes caused by the docking and undocking dynamics of the ribosomal protein S4-rRNA complex. (b) FRET histograms of the ribosomal protein S4-rRNA complex dynamics at 20 mm MgCl 2. The average dwell times on the high FRET state are 4.6 s for the PEG surface and 5.5 s for the DT20 surface while the average dwell time on the low FRET state is 1.5 s for both surfaces. Close agreements on the histograms and average dwell times indicate the unaltered dynamics on the DT20 surface.

13 Supplementary Figure 13 The dissociation curve of biotinylated BSA on the DT20 surface. Fluorophore-labeled biotinylated DNA was immobilized through NeutrAvidin, which was, in turn, immobilized through biotinylated BSA molecules on the surface. Any free fluorophore-labeled DNA was washed away after this immobilization step. Average surface spot counts over an imaging area of 2500 µm 2 were recorded at different time points up to 2 hours. To avoid the photobleaching issue, imaging buffer was used and the same area was never exposed more than once. The dissociation of biotinylated BSA from the surface can be monitored by the decrease in the surface spot counts of fluorophore-labeled DNA. Error bars indicate s.d.

14 Supplementary Figure 14 The durability of the Tween-20 passivation layer. Non-specific binding tests of 500 nm Cy3-labeled ssdna (34 nt) over an imaging area of 2500 µm 2 were conducted at different time points after the Tween-20 passivation treatment was done. Up to 5 hours after passivation, the Tween-20 layer is still effective. Error bars indicate s.d.

15 Supplementary protocol Preparation of the DDS-coated surface: 1) If one starts with a used flow chamber, it is necessary to remove the tape and epoxy by soaking the flow chamber in 1:1 acetone/methanol solution. 2) Clean the recycled quartz slides by scrubbing and sonicating in 2 % Alconox for 20 min. Then rinse with tap water. Make sure after this step, no tape or epoxy remains on the slides. 3) Rinse and boil the slides in MilliQ in a microwave oven for 10 min. Repeat this step twice to make sure the slides look clean. Additional Piranha and oxygen plasma cleaning can also be used. Do not put slides with tape or epoxy in Piranha solution! 4) Place the slides and coverslips in a glass slide holder. Rinse twice with MilliQ. 5) Rinse the slides and coverslips with acetone (Fisher Chemical, Certified ACS) once. Then sonicate in acetone for 20 min. 6) Rinse the slides and coverslips with methanol (BAKER ANALYZED, ACS Reagent) once. Then sonicate in methanol for 20 min. 7) Rinse the slides and coverslips with MilliQ once. Then sonicate in 1 M KOH for 1 hour. We mainly used KOH etching to clean and activate the surface. We also tried other methods, such as Piranha and oxygen plasma cleaning with similar results. Notice that prolonged burning is sometimes used to dry the surface. However, it may lead to dehydration of the surface hydroxyl groups. Therefore, the burning step should be kept brief or eliminated. 8) Rinse the slides and coverslips with MilliQ thoroughly. 9) Rinse the slides and coverslips with acetone once. Then sonicate in acetone for 20 min. 10) Rinse the slides and coverslips with methanol once. Then sonicate in methanol for 20 min.

16 11) Rinse the slides and coverslips with MilliQ once. Then sonicate in 5 M KOH for 1 hour. During this step, one need start to clean a polypropylene (PP, Bel-Art, Scienceware ) slide holder by rinsing and sonicating with hexane (Fisher Chemical, Spectranalyzed ) several times. Keep the PP slide holder away from contamination. A glass slide holder can also be used. 12) Rinse with the slides and coverslips MilliQ thoroughly. 13) Air-dry the PP slide holder thoroughly. Air-dry the slides and coverslips thoroughly and place them in the PP slide holder. 14) Rinse the slides and coverslips twice with hexane. Besides hexane, cyclohexane (AR (ACS), Macron Fine Chemicals ) is another suitable solvent we tested. 15) Add 75 ml hexane and ~50 L dichlorodimethylsilane (DDS, Aldrich, >99.5%) using a 1 ml syringe with needle. Put the needle tip under hexane to avoid air contact and inject DDS quickly since it is extremely reactive with moisture. 16) Immediately seal the holder tightly with its cap. Gently shake the holder at room temperature for 1.5 hours. 17) Dump the hexane solution into a designated hexane-dds waste bottle. 18) Rinse and sonicate the slides and coverslips in hexane for 1 min. Repeat this step for 3 times. 19) Air-dry the slides and coverslips. 20) Put one pair of slide and coverslip in a 50 ml falcon tube. Vacuum seal the tube in a food saver bag and store it at -20 C. Storage at -20 C can preserve the slides and coverslips for about 2 months. Using the DDS/Tween-20 slides: 1) Take the slides and coverslips out from -20 C and warm them up to room temperature. 2) Assemble them into flow chambers in the same manner as for the regular PEG slides.

17 3) Flow in 50 L 0.2 mg/ml biotinylated BSA (A8549, Sigma) in T50 buffer (20 mm Tris and 50 mm NaCl at ph 8.0) in each channel and incubate for 5 min. 4) Flow in 100 L 0.2 % Tween-20 (Fisher BioReagents ) in T50 buffer in each channel and incubate for 10 min. 5) The NeutrAvidin and sample solutions can be added in the same way as for the regular PEG slides. Note: A pure DDS/Tween-20 surface was made without embedded BSA/NeutrAvidin by skipping the steps of biotinylated BSA and NeutrAvidin incubation.