Super-Resolution Imaging of Chromosomal DNA in Cells

Abstract

Super-resolution imaging is achieved by localizing diffraction-limited spots with high accuracy. Here we combined two powerful approaches to image the chromosomal DNA inside cells. In one method, the accumulated, stochastic binding of fluorescent labels to an imaging target are localized, while in the other the fluorophore transitions between dark and bright states (compatible with binding, photobleaching, photo-activation, blinking, etc.), even when fluorophore images overlap, are localized. Combining the two techniques results in a robust microscopy that is faster than what is possible with either technique alone, requires less optimization, and corrects for cell autofluorescence. In addition, background noise due to fluorescent labels in solution can be virtually eliminated by using labels that fluoresce only when bound to the target. Many DNA-specific dyes show dramatic fluorescence enhancement upon binding to DNA, including SYTO, LOLO, and YOYO dyes. We used nanomolar concentrations of SYTO and LOLO to image lambda DNA attached to poly-L-lysine coated glass and chromosomal DNA in fixed HEK 293 and HeLa cells. We found average single-fluorophore localization errors of 36 nm and 24 nm on glass and in cells, respectively. These imaging techniques may prove useful in future studies of chromosomal DNA in cells, including chromatin structure and defects. This approach was further applied to imaging microtubules in vitro. We used commercial Oregon Green 488 paclitaxel to achieve a 10 nm average fluorophore localization error, and streptavidin S45A to transiently label biotinylated microtubules with Atto647N, resulting in an average of 18 nm fluorophore localization error. Future work will involve simultaneous imaging of DNA and proteins to answer important biological questions.