Improved method for counting DNA molecules on biofunctionalized nanoparticles.

In order to accurately determine low numbers (1-100) of immobilized ssDNA molecules at a single, silica 250 nm nanoparticle surface, we hereby propose an integrated approach combining classic single molecule confocal microscopy (SMCM), that is, stepwise photobleaching of labeled ssDNA, with modified total internal reflection fluorescence microscopy (mTIRF). We postulate that SMCM alone is unable to exactly account for all labeled ssDNA because of inherent laser polarization effects; that is, perpendicularly oriented molecules to the sample surface are not (or are only slightly) susceptible to laser excitation and thus are invisible in a classic photobleaching experiment. The SMCM method accounts for at best two-thirds (68%) of the present ssDNA molecules. The principle of the mTIRF technique, which relies on the creation of highly inclined illumination combined with part of the laser remaining in normal Kohler illumination, enables accurate counting of SMCM invisible molecules. The combined approach proposed here circumvents the polarization issue and allows a complete single molecule counting on individual nanoparticles, fully in line with bulk measurements, as will be demonstrated.