Molecular Counting with DNA Origami - Verification and Validation Towards Biological Applications

Single-molecule localization microscopy (SMLM) has been a powerful tool for expanding our understanding of cell biology. By taking advantage of the stochastic blinking that fluorophores naturally exhibit and controlling the dynamics of this process, SMLM pushes the level of resolution an order-of-magnitude beyond the diffraction limit allowing light microscopy to visualize cellular components with improved resolution. There is also tremendous interest in using the technique to count single molecules. The main challenge of molecular counting in SMLM is largely a result of the multiple and random blinking of fluorophores, which leads to over-counting the number of molecules. In [1], we proposed a method for counting biomolecules based on the blinking statistics of fluorophores and taking into account the labeling efficiency to the target molecule. We show how our theory may be used by analyzing simulated data [1], and in vitro data based on DNA origami structures [2]. Accurately determining the number of proteins or nucleic acids in a cell has wide-ranging applications, from systems biology, to proteomics/genomics, to fundamental cell biology. As a potential application, we’ve begun to apply this technique to the problem of accurately determining plasmid copy number in individual bacteria.