Revealing Spatial\nMolecular Heterogeneity of High-Density\nBiofunctionalized Surfaces Using DNA-PAINT

The quantification and control of molecular densities\nand distributions\non biofunctionalized surfaces are key for enabling reproducible functions\nin biosciences. Here, we describe an analysis methodology for quantifying\nthe density and spatial distribution of high-density biofunctionalized\nsurfaces, with densities in the order of 10<sup>2</sup>–10<sup>5</sup> biomolecules per μm<sup>2</sup> area, in a short measurement\ntime. The methodology is based on single-molecule DNA-PAINT imaging\ncombined with simulation models that compensate for lifetime and spatial\nundersampling effects, resulting in three distinct molecule counting\nmethods and a statistical test for spatial distribution. The analysis\nmethodology is exemplified for a surface with ssDNA affinity binder\nmolecules coupled to a PLL-<i>g</i>-PEG antifouling coating.\nThe results provide insights into the biofunctionalization efficiency,\nyield, and homogeneity. Furthermore, the data reveal that heterogeneity\nis inherent to the biofunctionalization process and shed light on\nthe underlying molecular mechanisms. We envision that DNA-PAINT imaging\nwith the developed analysis framework will become a versatile tool\nto study spatial heterogeneity of densely biofunctionalized surfaces\nfor a wide range of applications.