“…The FSF procedure described above was modified to include a preembedding gold-enlarging technique for HPF cells by adapting approaches that involve silver or gold enhancement at room temperature (Danscher, 1981;Hacker et al, 1988;Hainfeld and Furuya, 1995;Scopsi, 1989), gold-toning (Sawada and Esaki, 2000), seed-mediated gold-enlarging (Busbee et al, 2003;Daniel and Astruc, 2004;Gole and Murphy, 2004;Handley, 1989;Jana et al, 2001;Meltzer et al, 2001;Okitsu et al, 2005;Zou et al, 2006) and a FSF-based silver-enhancement procedure (Morphew et al, 2007). To avoid the background that results from spontaneous autonucleation, we designed a three-step enlarging protocol in which silver enhancement was used to slightly enlarge the Nanogold, the silver shell was coated by gold toning to make it insoluble in osmium, and the particles were further enlarged to 10 -16 nm using gold enhancement.…”