Multiplexed Super-Resolution Imaging using Programmable DNA-based BarcodesView all posters
Rice University, United States
Synthetic biologists have engineered biological circuits to perturb increasingly complex systems such as eukaryotic cells. To better understand these processes, it is necessary to visualize the spatial distributions of many proteins with high resolution. Super-resolution microscopy techniques such as STORM have enabled structures smaller than 20nm to be resolved. However, these techniques are inherently limited by the number of targets that can be visualized in one sample. Currently, no immunofluorescent super-resolution approach has been able to image more than six protein targets because of signal loss associated with cross-talk between spectrally similar fluorophores. Here, we demonstrate a cost-effective technique to overcome this limitation in fixed cells by encoding many protein targets with temporal DNA-based barcodes. These barcodes use dynamic DNA complexes. We can selectively label and remove fluorescent signals by utilizing programmable sequence-specific strand displacement reactions. These reactions occur between target-bound DNA-conjugated primary antibodies and STORM dye pair-conjugated DNA complexes. Then, we re-iteratively localize each individual target molecule with a permuted fluorescent signal in sequential imaging rounds. By doing so, we are able to encode each target with a color-based temporal barcode. Importantly, the number of targets that can be imaged using this technique scales polynomially (the number of fluorescent channels to the power of the number of imaging rounds). Here, we show that DNA-based temporal barcodes can be used to image ten or more immunofluorescent targets at super-resolution.