Supplementary Figure 10: Live cell super-resolution imaging of RNA aptamers.

(a)-(c), Photostability of different RMFPs and FPs in live 293T/17 cells. (a) Cells expressing Pepper, Broccoli, mCherry-H2B, mVenus-H2B, UnaG-H2B, EGFP-H2B, or ECFP-H2B were imaged using a confocal laser scanning microscope with a 561 nm laser for Pepper599, Pepper620 and mCherry, a 488 nm laser for Pepper525, Pepper530, mVenus-H2B, UnaG-H2B and EGFP-H2B, a 458 nm laser for Pepper514, Pepper508, Pepper497, Pepper485, Broccoli-DFHBI-1T and ECFP-H2B. The curves were normalized to spectra of the RNA-fluorophore complexes or fluorescent proteins. (b) The same cells in (a) were imaged using a 1040 nm two-photon excitation for Pepper599, Pepper620 and mCherry, a 976 nm two-photon excitation for Pepper525, Pepper530, mVenus-H2B, UnaG-H2B and EGFP-H2B, a 900 nm two-photon excitation for Pepper514, Pepper508, Pepper497, Pepper485, Broccoli-DFHBI-1T and ECFP-H2B. (c) Photoswitching properties of Broccoli, Pepper497, Pepper508, Pepper 514, Pepper525, Pepper530 in live cells. Cells expressing Pepper or Broccoli were labeled by their cognate ligands and imaged with five cycles of continuous 458 nm or 488 nm laser scanning for 10 seconds (solid lines in the white regions), with 60 seconds of darkness between cycles (gray regions). The percent recovery (%REC) of the peak initial fluorescence was almost 100% for both Broccoli and Peppers, for according to the equation: \(\% REC = \frac{{f_r - f_{bl}}}{{f_0 - f_{bl}}}\), where f₀ is the peak initial fluorescence, f_bl is the post-bleach fluorescence, and f_r is the post-dark recovery fluorescence⁸. All data in (a-c) are normalized to the initial image intensity (at time 0), N=10 cells. (d-n) HeLa cells expressing Pepper tethered to H2B in nuclei (d-g) or TOMM20 on the outer mitochondrial membrane (k-m) were studied by widefield and 3D-SIM imaging in the presence of HBC620. The lateral and axial spatial resolutions of SIM imaging were 116-nm and 350-nm, respectively. (d, k) Wide-field imaging showing co-localization of BFP and Pepper620. (e) Apical section taken from the surface of the nuclear envelope close to the coverslip. (f) Images of H2B-tethered Pepper in mid-cross-section. 3D-SIM images reveal a punctated pattern of regions devoid of Pepper620 fluorescence. (g) An x-z planar cross-section through the dashed line shown in (f). (h-j) Raw images of Broccoli-DFHBI-1T, Pepper530 and Pepper620 for SIM imaging. HeLa cells expressing 2Broccoli-MS2 (h) or 4Pepper-MS2 and tdMCP-tagBFP-H2B (i, j) were imaged by structured illumination microscopy (SIM) imaging in the presence of 20 μM DFHBI-1T (h), 2 μM HBC (i) or 1 μM HBC620 (j). (l) Images of TOMM20-tethered Pepper shown in maximum-intensity projection along the z dimension through the cell volume. (m) One x–z planar cross-section through the dashed line shown in (l). (n) 3D SIM versus conventional wide-field with depth encoded in color. This `ure shows the maximum-intensity projection of a HeLa cell expressing 4Pepper-MS2 and TOMM20-tdMCP-tagBFP, and is mostly the same as (l) except that different colors are used to indicate the axial position, or depth, of the Pepper620. The depth-to-color map is shown at the bottom right corner. Scale bars in (d-f), 5 μm; (h-l) and (n), 10 μm; (g) and (m), 1 μm. For d-n, at least two independent experiments were carried out with similar results.