Single-fluorescent oligonucleotide molecules hybridized to functionalized biochips. (A) Surface functionalized with probe oligonucleotide upon hybridization with 100 fM of Cy5-labeled target oligonucleotide. Individual diffraction-limited peaks, corresponding to single oligonucleotide molecules, can be resolved at a surface density of 0.8 μm−2. (B) Surface functionalized with control sequence yielded a 1000-fold lower signal compared with specific hybridization. We ensured that the control can bind to a complementary strand by hybridizing with 60-pM complementary fluorescent (Cy5-labeled) Xenopus XAG2-cDNA (Supplemental Fig. S1). (C) The high single-molecule brightness of 2600 counts and the low background noise of 34 counts allow reliable detection of more than 99% of all surface-bound dye molecules.
