Proteins in cells assemble into nanoscale complexes in order to carry out a specific function. The spatial organization and stoichiometry of proteins within these nanoscopic functional units is highly important for maintaining a cell’s healthy physiology. Changes in nanoscale organization of protein complexes, for example a change from monomeric to oligomeric stoichiometry, often triggers disease states. However, visualizing many proteins simultaneously and quantifying protein copy number with high spatial resolution is highly challenging. Super-resolution methods hold promise for overcoming this hurdle, however, the complex photophysics of fluorophores limits both multi-color imaging capabilities and the ability to extract quantitative information. To address the challenges with quantification of protein copy number at the nanoscale level, we built nanotemplates and calibration standards based on DNA origami. These calibration standards have allowed us to extract the copy number of small protein assemblies including motor protein like dynein.

One major limitation of existing SMLM methods is the inability to image many cellular components with high throughput. Stochastic Optical Reconstruction Microscopy (STORM) is one popular SMLM method, but we showed that only one photoswitchable fluorophore used in STORM (AF647) provides high quality images of many biological structures in particular nuclear structures, dramatically limiting multicolor capability. DNA-Point accumulation for imaging in nanoscale topography (DNA-Paint) is another popular SMLM method that uses conventional rather than photoswitchable fluorophores overcoming limitations associated to multi-color STORM imaging. However, multi-color DNA-Paint is typically performed sequentially and the slow imaging speed compromises imaging throughput. To overcome this challenge, we developed a new multi-color DNA-Paint approach that utilizes fluorophore absorption rather than emission to discriminate color and that relies on frequency multiplexing of the laser excitation. We call this approach frequency multiplexed (fm) DNA-Paint. fm-DNA-Paint has the potential to image up to 5 colors in one shot in the same amount of time as conventional single color DNA-Paint.