Accelerated super-resolution imaging with FRET-PAINT

We first tested the feasibility of FRET-PAINT microscopy using surface-immobilized DNA strands and a total internal reflection fluorescence (TIRF) microscope. In the scheme of FRET-PAINT, three DNA strands—docking, donor, and acceptor strands—are used (Fig. 1a). The docking strand (Docking_P0, Additional file 1) labeled with a biotin at the 5’-end has two docking sites, each of which base-pairs with the donor or acceptor strand. To maintain the photobleaching resistance and high multiplexing capability of DNA-PAINT, both the donor and acceptor strands should be easily replenished with new ones. We used 9 nt donor and 10 nt acceptor strands, which have dissociation rates of 1.2 Hz and 0.02 Hz, respectively (Additional file 1: Figure S1). In this way, photobleached donor and acceptor strands can be continuously replenished by those in the solution. To increase the FRET probability upon donor strand’s binding to the docking strand, we chose a shorter length for the donor strand than for the acceptor strand whereas relatively longer acceptor strand was used. Therefore, the switching speed of FRET-PAINT in our scheme is mainly determined by the dissociation rate of the donor strand. Compared to DNA-PAINT, the length of the docking strand of FRET-PAINT is increased a little bit, but the additional position uncertainty induced by the increased docking strand length is just a few nanometers, which is negligible in most biological applications.

The donor strand was labeled at the 3’-end with Alexa488 (Donor_P1_Alexa488, Additional file 1) whereas the acceptor strand was labeled with Cy5 (Acceptor_P11_Cy5, Additional file 1) at the 3’-end. We immobilized the docking strand on a polymer-coated quartz surface using a streptavidin-biotin interaction and took single-molecule images of Cy5 after injecting the donor (1000 nM) and acceptor (100 nM) strands by exciting Alexa488 using a blue laser. In the scheme, we do not directly excite Cy5 (Fig. 1b), and therefore we could use such high concentrations of donor and acceptor strands without worrying about background noise. As Fig. 1c shows, we obtained clear Cy5 fluorescence intensity time traces at such high donor and acceptor concentrations.

Using the same scheme, we tried to find the optimum labeling position of FRET probes that gives maximum FRET signal for two FRET pairs: Cy3-Cy5 and Alexa488-Cy5. The general requirements for FRET pairs are large spectral overlap between donor emission and acceptor excitation for high FRET efficiency and small spectral overlap between donor emission and acceptor emission for low background noise. The Cy5 was chosen as an acceptor due to its superior photophysical properties such as high photostability and brightness. Alexa488 and Cy3 were selected as a donor because they are photostable and their fluorescence spectra do not significantly overlap with that of Cy5. We prepared donor strands labeled with either Cy3 (Donor_P1_Cy3, Additional file 1) or Alex488 (Donor_P1_Alexa488) at the 3’-end, and acceptor strands labeled with Cy5 at varying positions (Acceptor_P2_Cy5, P4_Cy5, P6_Cy5, and P11_Cy5, Additional file 1). The Cy3-Cy5 FRET pair gave the highest Cy5 signal when the gap between donor and acceptor fluorophores was 6 nt, whereas Alexa488-Cy5 FRET pair gave the highest Cy5 signal when the gap was 2 nt (Fig. 1d). We used these optimized labeling schemes for the remaining part of the paper.

In super-resolution fluorescence imaging, HILO (Highly Inclined and Laminated Optical sheet) [17] microscopy is conventionally used. We compared signal-to-noise ratios (SNRs) of DNA-PAINT and FRET-PAINT at varying DNA concentrations in HILO setup. Figure 1e is DNA-PAINT images of surface immobilized docking strand (Docking_P0) at varying imager strand (Acceptor_P11_Cy3, Additional file 1) concentrations. Single-molecule images started to be overwhelmed by background noise when image concentration was above 5 nM. Figure 1f is FRET-PAINT images of the docking strand at varying donor (Donor_P1_Cy3) concentrations with acceptor (Acceptor_P6_Cy5) concentration fixed at 10 nM. Figure 1g is FRET-PAINT images of the docking strand at varying acceptor (Acceptor_P6_Cy5) concentrations with donor (Donor_P1_Cy3) concentration fixed at 10 nM and. Figure 1h is FRET-PAINT images of the docking strand at varying donor (Donor_P1_Alexa488) concentrations with acceptor (Acceptor_P2_Cy5) concentration fixed at 10 nM. Figure 1i is FRET-PAINT images of the docking strand at varying acceptor (Acceptor_P2_Cy5) concentrations with donor (Donor_P1_Alexa488) concentration fixed at 10 nM. These images clearly show that similar SNR can be obtained at much higher imager concentrations in FRET-PAINT compared to DNA-PAINT. For instance, we used 5 nM imager concentration for DNA-PAINT to obtain the 3.3 SNR (Fig. 1j, k). For the same SNR, we could use 180 nM donor and 120 nM acceptor concentrations for the Cy3-Cy5 pair, and 250 nM donor and 90 nM acceptor concentrations for the Alexa488-Cy5 pair, respectively (Fig. 1j, k). Exact sample numbers for these analyses are described in the Methods section and the error bars represent standard error.

Next we compared the superresolution imaging speeds of DNA-PAINT and FRET-PAINT. Microtubules of COS-7 cells were immunostained with the anti-tubulin antibody which is labeled with Docking_P1 (Additional file 1). For DNA-PAINT, microtubules were imaged after injecting 1 nM Cy5-labeled imager strand (Acceptor_P2’_Cy5). Single-molecule images were recorded at a frame rate of 10 Hz, which is fast enough to reliably detect binding of donor and acceptor strands (Additional file 1: Figure S1). Figure 2a shows super-resolution images reconstructed at varying acquisition times. Since 18000 frames in total were recorded at a frame rate of 10 Hz for Fig. 2a, the total imaging time was 30 min. For FRET-PAINT, microtubules of the same area were imaged after injecting 30 nM donor (Donor_P1_Alexa488) and 20 nM acceptor (Acceptor_P2_Cy5) strands. Figure 2b shows super-resolution images reconstructed at varying acquisition times. Since 600 frames were recorded at a frame rate of 10 Hz, total imaging time was 60 s. Even by simple eye inspection, it is clear that the speed of FRET-PAINT is much faster than that of DNA-PAINT. To quantitatively compare the imaging speed of DNA-PAINT and FRET-PAINT, we first measured the number of localized spots of Fig. 2a-b as a function of imaging time, and observed 29-fold increase of the imaging speed (Fig. 2c). The same analysis was performed for nine additional imaging areas, and the averaged results are summarized in Fig. 2d, revealing 32-fold increase of the imaging speed on average. As a second way to compare the imaging speeds of DNA-PAINT and FRET-PAINT, we quantified the convolved resolutions computed as ((localization precision)² + (Nyquist resolution)²)^1/2 as previously reported (Fig. 2e) [18]. In our experiments, the localization precisions of DNA-PAINT and FRET-PAINT were 6.9 nm and 11.1 nm, respectively (Additional file 1: Figure S2). Because we used the same excitation laser powers for DNA-PAINT and FRET-PAINT, we think that the little difference in localization precision was caused by the difference in the extinction coefficients of Alexa488 and Cy5. As Fig. 2e shows, the convolved resolution was nicely fitted to a linear function in the log-log plot, indicating that the resolution is mainly determined by Nyquist resolution, which is inversely proportional to the square root of the localization density [19]. By comparing the y-intercept of the two fitting lines, we obtained 36-fold increase of imaging speed.

Finally, we assessed the multiplexing capability of FRET-PAINT microscopy. Microtubules and mitochondria of COS-7 cells were immunostained using anti-tubulin antibody and anti-Tom20 antibody, respectively. The anti-tubulin antibody and anti-Tom20 antibody were orthogonally conjugated with Docking_P1 and Docking_P2 (Additional file 1), respectively. Two different approaches were used for multiplexed imaging. In the one approach (Fig. 3a), we imaged microtubules first by injecting 20 nM Donor_P1_Alexa488 and 10 nM Acceptor_P2_Cy5 (Fig. 3b) and then imaged mitochondria by injecting 10 nM Donor_P2_Alexa488 (Additional file 1) and 10 nM Acceptor_P2_Cy5 (Fig. 3c). Figure 3d shows an overlaid image of Fig. 3b and Fig. 3c. Spatial organization of microtubules and mitochondria are clearly visualized. In the second approach (Fig. 3e), we injected all DNA probes (10 nM Donor_P1_Cy3 for microtubules, 20 nM Donor_P2_Alexa488 for mitochondria, and 10 nM Acceptor_P2’_Cy5 for both microtubules and mitochondria, Additional file 1) at the same time, and imaged microtubule first with Cy3 excitation (Fig. 3f), and then imaged mitochondria with Alexa488 excitation (Fig. 3g). Figure 3h shows an overlaid image of Fig. 3f and Fig. 3g. Even though the second approach has no advantage in terms of the imaging time, its experimental time was actually decreased because no buffer exchange is required. A disadvantage of the second approach is a cross-talk between microtubule and mitochondria images (Additional file 1: Figure S3). Even though the cross-talk could be partially removed by using intensity filtering, a significant amount of mitochondria were lost during intensity filtering (Additional file 1: Figure S3), demonstrating that the sequential imaging scheme is a better way to do multiplexing imaging.

Modified DNA oligonucleotides were purchased from Integrated DNA Technologies. Alexa488 (Alexa Fluor 488 NHS Ester, catalog number: A20000) was purchased from Thermo Fisher Scientific. Cy3 (Cy3 NHS Ester, catalog number: PA13101) and Cy5 (Cy5 NHS Ester, catalog number: PA15101) were purchased from GE Healthcare Life Sciences. COS-7 cells were purchased from Korean Cell Line Bank. Anti-tubulin antibody (catalog number: ab6160) was purchased from Abcam. Anti-Tom20 antibody (sc-11415) was purchased from Santa Cruz Biotechnology, Inc. Donkey anti-rabbit IgG antibody (catalog number: 711-005-152) and donkey anti-rat IgG antibody (catalog number: 712-005-153) were purchased from Jackson ImmunoResearch Laboratories, Inc. Carboxyl latex beads (catalog number: C37281) were purchased from Thermo Fisher Scientific. The docking strands were conjugated to the secondary antibodies using Antibody-Oligonucleotide All-in-One Conjugation Kit (catalog number: A-9202-001) purchased from Solulink. Paraformaldehyde (catalog number: 1.04005.1000) was purchased from Merck. Glutaraldehyde (catalog number: G5882), Triton X-100 (catalog number: T9284), and Bovine Serum Albumin (catalog number: A4919) were purchased from Sigma-Aldrich.

Amine-modified DNA oligonucleotides were labeled with fluorophores which have NHS ester chemical group. 5 ul of 1 mM DNA was mixed with 25 ul of 100 mM sodium tetraborate buffer (pH 8.5). And then 5ul of 20 mM fluorophore in DMSO was added. After thorough mixing, the mixture was incubated at 4°C overnight while protected from light. 265 ul of distilled water, 900 ul of ethanol, and 30 ul of 3 M sodium acetate (pH 5.2) were added and mixed thoroughly. The mixture was incubated at -20°C for an hour and then centrifuged for a couple of hours until the DNA pellet is clearly visible. Supernatant was discarded and the pellet was washed with cold ethanol. After ethanol was evaporated completely, the pellet was resuspended in 50 ul of distilled water and the labeling efficiency was measured. If the labeling efficiency is low, the whole labeling process was repeated.

For drift correction of DNA-PAINT imaging, #1.5 glass coverslips were sparsely coated with carboxyl latex beads. The coverslip was coated with bead solution 1:10 diluted in distilled water, heated for 10 minutes on a 100°C hot plate, washed thoroughly with distilled water, and dried with N2 gas. COS-7 cells were grown on bead-coated coverslips for a few days and then fixed for 10 minutes. 2% glutaraldehyde in cytoskeleton buffer was used for microtubule imaging (Fig. 2) and 3% paraformaldehyde and 0.1% glutaraldehyde mixture in PBS buffer was used for microtubule and mitochondria imaging (Fig. 3) [26, 27]. Fixed samples were stored at 4°C in PBS buffer until needed. A flow channel was made by assembling the cell-covered coverslip and a glass slide using double-sided tape and epoxy. In the glass slide, two holes were pre-made for convenient buffer exchange.

Microtubules were immunostained by injecting 1:100 diluted anti-tubulin antibody in blocking solution (5% Bovine Serum Albumin and 0.25% Triton X-100 in PBS buffer) into the channel and incubating at 4°C overnight. After thorough wash-out of free anti-tubulins with PBS buffer, cells were incubated with 100 nM secondary antibody conjugated with docking strand (Docking_P1, Additional file 1) for 1 hour. Mitochondria were immunostained by injecting 1:100 diluted anti-Tom20 antibody in blocking solution into the channel and incubating at 4°C overnight. After thorough wash-out of free anti-Tom20 antibody with PBS buffer, cells were incubated with 100 nM secondary antibody conjugated with docking strand (Docking_P2) for 1 hour.

For single-molecule imaging, a prism-type total internal reflection fluorescence (TIRF) microscopy and highly inclined and laminated optical sheet (HILO) microscopy were used. The microscope was built by modifying a commercial inverted microscope (IX71, Olympus), and equipped with a 100X 1.4 NA oil-immersion objective lens (UPlanSApo, Olympus). To obtain data in Fig. 1, docking strands were immobilized on the polymer-coated quartz slide surface by using streptavidin-biotin interaction, and donor and acceptor strands were added into the imaging channel. Alexa488, Cy3, and Cy5 were excited by a blue laser (473 nm, 100 mW, MBL-III-473-100mW, CNI), a green laser (532 nm, 50 mW, Compass 215M-50, Coherent), and a red laser (642 nm, 60 mW, Excelsior-642-60, Spectra-Physics), respectively. Cy3 signal was filtered using a dichroic mirror (640dcxr, Chroma), and Cy5 signal was filtered using a dichroic mirror (740dcxr, Chroma). Single-molecule images were recorded at a frame rate of 10 Hz with electron multiplying charge coupled device (EMCCD) camera (iXon Ultra DU-897U-CS0-#BV, Andor).

To characterize detected photons per frame in Fig. 1d, 13997 (8096), 11021 (5100), 11208 (3451), and 17051 (3980) single-molecule spots were collected for 2 nt, 4 nt, 6 nt, and 11 nt Cy3-Cy5 (Alexa488-Cy5) FRET pairs, respectively. To characterize SNR in Fig. 1j-k, 795, 2322, and 742 single-molecule spots were collected for Cy3, Cy3-Cy5 pair, and Alexa488-Cy5 pair, respectively.

For super-resolution imaging with DNA-PAINT, we used a home-made auto-focusing and drift correction system based on image correlation method. Before filming, one in-focus bright field image and two out-of-focus images were taken. These three reference images were used to keep track of x, y, and z axes drift [28]. The drift in z-direction was corrected in real time using a piezo stage (PZ-2000, Applied Scientific Instrumentation) whereas the drift in x-y plane was corrected during image analysis.